Antibosies to PDGF-D

ABSTRACT

PDGF-D, a new member of the PDGF/VEGF family of growth factors, as well as the nucleotide sequence encoding it, methods for producing it, antibodies and other antagonists to it, transfected and transformed host cells expressing it, pharmaceutical compositions containing it, and uses thereof in medical and diagnostic applications, including methods for stimulating growth of a connective tissue or healing a wound in a mammal, which methods comprise administering to the mammal an effective amount of PDGF-D polypeptides or polynucleotides encoding the PDGF-D polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/260,539,filed Oct. 1, 2003, which in turn is a continuation-in-part of U.S.application Ser. No. 10/086,623, filed Mar. 4, 2002, which is acontinuation-in-part of U.S. application Ser. No. 09/691,200, filed Oct.19, 2000, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 09/438,046, filed Nov. 10, 1999, now U.S. Pat. No.6,706,687, and claims the benefit of U.S. Provisional Application No.60/107,852, filed Nov. 10, 1998; U.S. Provisional Application No.60/113,997, filed Dec. 28, 1998; U.S. Provisional Application No.60/150,604, filed Aug. 26, 1999; U.S. Provisional Application No.60/157,108, filed Oct. 4, 1999; and U.S. Provisional Application No.60/157,756, filed Oct. 5, 1999.

FIELD OF THE INVENTION

This invention relates to growth factors for cells expressing receptorsto a novel growth factor that include endothelial cells, connectivetissue cells (such as fibroblasts) myofibroblasts and glial cells, andin particular to a novel platelet-derived growth factor/vascularendothelial growth factor-like growth factor, polynucleotide sequencesencoding the factor, and to pharmaceutical and diagnostic compositionsand methods utilizing the factor for stimulating connective tissuegrowth or promoting wound healing.

BACKGROUND OF THE INVENTION

In the developing embryo, the primary vascular network is established byin situ differentiation of mesodermal cells in a process calledvasculogenesis. It is believed that all subsequent processes involvingthe generation of new vessels in the embryo and neovascularization inadults, are governed by the sprouting or splitting of new capillariesfrom the pre-existing vasculature in a process called angiogenesis(Pepper et al., 1996, Enzyme & Protein, 49:38-162; Breier et al., 1995,Dev. Dyn., 204:228-239; Risau, 1997, Nature, 386:671-674). Angiogenesisis not only involved in embryonic development and normal tissue growth,repair, and regeneration, but is also involved in the femalereproductive cycle, establishment and maintenance of pregnancy, and inrepair of wounds and fractures. In addition to angiogenesis which takesplace in the normal individual, angiogenic events are involved in anumber of pathological processes, notably tumor growth and metastasis,and other conditions in which blood vessel proliferation, especially ofthe microvascular system, is increased, such as diabetic retinopathy,psoriasis and arthropathies. Inhibition of angiogenesis is useful inpreventing or alleviating these pathological processes.

On the other hand, promotion of angiogenesis is desirable in situationswhere vascularization is to be established or extended, for exampleafter tissue or organ transplantation, or to stimulate establishment ofcollateral circulation in tissue infarction or arterial stenosis, suchas in coronary heart disease and thromboangitis obliterans.

The angiogenic process is highly complex and involves the maintenance ofthe endothelial cells in the cell cycle, degradation of theextracellular matrix, migration and invasion of the surrounding tissueand finally, tube formation. The molecular mechanisms underlying thecomplex angiogenic processes are far from being understood.

Because of the crucial role of angiogenesis in so many physiological andpathological processes, factors involved in the control of angiogenesishave been intensively investigated. A number of growth factors have beenshown to be involved in the regulation of angiogenesis; these includefibroblast growth factors (FGFs), platelet-derived growth factor (PDGF),transforming growth factor alpha (TGFα), and hepatocyte growth factor(HGF). See for example Folkman et al., 1992, J. Biol. Chem.,267:10931-10934 for a review.

It has been suggested that a particular family of endothelialcell-specific growth factors, the vascular endothelial growth factors(VEGFs), and their corresponding receptors are primarily responsible forstimulation of endothelial cell growth and differentiation, and forcertain functions of the differentiated cells. These factors are membersof the PDGF family, and appear to act primarily via endothelial receptortyrosine kinases (RTKs).

Eight different proteins have been identified in the PDGF family, namelytwo PDGFs (A and B), VEGF and five members that are closely related toVEGF. The five members closely related to VEGF are: VEGF-B, described inInternational Patent Application PCT/US96/02957 (WO 96/26736) whichcorresponds to U.S. Pat. No. 5,928,939, and in U.S. Pat. Nos. 5,840,693and 5,607,918 to Ludwig Institute for Cancer Research and The Universityof Helsinki; VEGF-C or VEGF-2, described in Joukov et al., 1996, EMBOJ., 15:290-298 and Lee et al., 1996, Proc. Natl. Acad. Sci. USA,93:1988-1992, and U.S. Pat. Nos. 5,932,540, 5,935,820 and 6,040,157;VEGF-D, described in International Patent Application No. PCT/US97/14696(WO 98/07832), and Achen et al., 1998, Proc. Natl. Acad. Sci. USA,95:548-553; the placenta growth factor (PlGF), described in Maglione etal., 1991, Proc. Natl. Acad. Sci. USA, 88:9267-9271; and VEGF3,described in International Patent Application Nos. PCT/US95/07283 (WO96/39421) and PCT/US99/18054 (WO 00/09148) by Human Genome Sciences,Inc. Each VEGF family member has between 30% and 45% amino acid sequenceidentity with VEGF. The VEGF family members share a VEGF homology domainwhich contains the six cysteine residues which form the cysteine knotmotif Functional characteristics of the VEGF family include varyingdegrees of mitogenicity for endothelial cells, induction of vascularpermeability and angiogenic and lymphangiogenic properties.

Vascular endothelial growth factor (VEGF) is a homodimeric glycoproteinthat has been isolated from several sources. VEGF shows highly specificmitogenic activity for endothelial cells. VEGF has important regulatoryfunctions in the formation of new blood vessels during embryonicvasculogenesis and in angiogenesis during adult life (Carmeliet et al.,1996, Nature, 380:435-439; Ferrara et al., 1996, Nature, 380:439-442;reviewed in Ferrara and Davis-Smyth, 1997, Endocrine Rev., 18:4-25). Thesignificance of the role played by VEGF has been demonstrated in studiesshowing that inactivation of a single VEGF allele results in embryoniclethality due to failed development of the vasculature (Carmeliet etal., 1996, Nature, 380:435-439; Ferrara et al., 1996, Nature,380:439-442). In addition VEGF has strong chemoattractant activitytowards monocytes, can induce the plasminogen activator and theplasminogen activator inhibitor in endothelial cells, and can alsoinduce microvascular permeability. Because of the latter activity, it issometimes referred to as vascular permeability factor (VPF). Theisolation and properties of VEGF have been reviewed; see Ferrara et al.,1991, J. Cellular Biochem., 47:211-218 and Connolly, J., 1991, CellularBiochem., 47:219-223. Alterative mRNA splicing of a single VEGF genegives rise to five isoforms of VEGF.

VEGF-B has similar angiogenic and other properties to those of VEGF, butis distributed and expressed in tissues differently from VEGF. Inparticular, VEGF-B is very strongly expressed in heart, and only weaklyin lung, whereas the reverse is the case for VEGF. This suggests thatVEGF and VEGF-B, despite the fact that they are co-expressed in manytissues, may have functional differences.

VEGF-B was isolated using a yeast co-hybrid interaction trap screeningtechnique by screening for cellular proteins which might interact withcellular retinoid acid-binding protein type I (CRABP-I). Its isolationand characteristics are described in detail in PCT/US96/02957 and inOlofsson et al., 1996, Proc. Natl. Acad. Sci. USA, 93:2576-2581.

VEGF-C was isolated from conditioned media of the PC-3 prostateadenocarcinoma cell line (CRL1435) by screening for ability of themedium to produce tyrosine phosphorylation of the endothelialcell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cellstransfected to express VEGFR-3. VEGF-C was purified using affinitychromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNAlibrary. Its isolation and characteristics are described in detail inJoukov et al., 1996, EMBO J., 15:290-298.

VEGF-D was isolated from a human breast cDNA library, commerciallyavailable from Clontech, by screening with an expressed sequence tagobtained from a human cDNA library designated “Soares Breast 3NbHBst” asa hybridization probe (Achen et al., 1998, Proc. Natl. Acad. Sci. USA,95:548-553). Its isolation and characteristics are described in detailin International Patent Application No. PCT/US97/14696 (WO98/07832).

The VEGF-D gene is broadly expressed in the adult human, but iscertainly not ubiquitously expressed. VEGF-D is strongly expressed inheart, lung and skeletal muscle. Intermediate levels of VEGF-D areexpressed in spleen, ovary, small intestine and colon, and a lowerexpression occurs in kidney, pancreas, thymus, prostate and testis. NoVEGF-D mRNA was detected in RNA from brain, placenta, liver orperipheral blood leukocytes.

PlGF was isolated from a term placenta cDNA library. Its isolation andcharacteristics are described in detail in Maglione et al., 1991, Proc.Natl. Acad. Sci. USA, 88:9267-9271. Presently its biological function isnot well understood.

VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3is stated to have about 36% identity and 66% similarity to VEGF. Themethod of isolation of the gene encoding VEGF3 is unclear and nocharacterization of the biological activity is disclosed.

Similarity between two proteins is determined by comparing the aminoacid sequence and conserved amino acid substitutions of one of theproteins to the sequence of the second protein, whereas identity isdetermined without including the conserved amino acid substitutions.

PDGF/VEGF family members act primarily by binding to receptor tyrosinekinases. Five endothelial cell-specific receptor tyrosine kinases havebeen identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3(Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosinekinase activity which is necessary for signal transduction. Theessential, specific role in vasculogenesis and angiogenesis of VEGFR-1,VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targetedmutations inactivating these receptors in mouse embryos.

The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1,VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity,and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be theligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., 1996,The EMBO Journal, 15:290-298). VEGF-D binds to both VEGFR-2 and VEGFR-3.A ligand for Tek/Tie-2 has been described in International PatentApplication No. PCT/US95/12935 (WO 96/11269) by RegeneronPharmaceuticals, Inc. The ligand for Tie has not yet been identified.

Recently, a novel 130-135 kDa VEGF isoform specific receptor has beenpurified and cloned (Soker et al., 1998, Cell, 92:735-745). The VEGFreceptor was found to specifically bind the VEGF₁₆₅ isoform via the exon7 encoded sequence, which shows weak affinity for heparin (Soker et al.,1998, Cell, 92:735-745). Surprisingly, the receptor was shown to beidentical to human neuropilin-1 (NP-1), a receptor involved in earlystage neuromorphogenesis. PlGF-2 also appears to interact with NP-1(Migdal et al., 1998, J. Biol. Chem., 273:22272-22278).

VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelialcells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia(Oelrichs et al., 1992, Oncogene, 8:11-18; Kaipainen et al., 1993, J.Exp. Med., 178:2077-2088; Dumont et al., 1995, Dev. Dyn., 203:80-92;Fong et al., 1996, Dev. Dyn., 207:1-10) and VEGFR-3 is mostly expressedin the lymphatic endothelium of adult tissues (Kaipainen et al., 1995,Proc. Natl. Acad. Sci. USA, 9:3566-3570). VEGFR-3 is also expressed inthe blood vasculature surrounding tumors.

Disruption of the VEGFR genes results in aberrant development of thevasculature leading to embryonic lethality around midgestation. Analysisof embryos carrying a completely inactivated VEGFR-1 gene suggests thatthis receptor is required for functional organization of the endothelium(Fong et al., 1995, Nature, 376:66-70). However, deletion of theintracellular tyrosine kinase domain of VEGFR-1 generates viable micewith a normal vasculature (Hiratsuka et al., 1998, Proc. Natl. Acad.Sci. USA, 95:9349-9354). The reasons underlying these differences remainto be explained but suggest that receptor signalling via the tyrosinekinase is not required for the proper function of VEGFR-1. Analysis ofhomozygous mice with inactivated alleles of VEGFR-2 suggests that thisreceptor is required for endothelial cell proliferation, hematopoesisand vasculogenesis (Shalaby et al., 1995, Nature, 376:62-66; Shalaby etal., 1997, Cell, 89:981-990). Inactivation of VEGFR-3 results incardiovascular failure due to abnormal organization of the large vessels(Dumont et al., 1998, Science, 282:946-949).

Although VEGFR-1 is mainly expressed in endothelial cells duringdevelopment, it can also be found in hematopoetic precursor cells duringearly stages of embryogenesis (Fong et al., 1995, Nature, 376:66-70). Itis also is expressed by most, if not all, vessels in embryos (Breier etal., 1995, Dev. Dyn., 204:228-239; Fong et al., 1996, Dev. Dyn.,207:1-10). In adults, monocytes and macrophages also express thisreceptor (Barleon et al., 1996, Blood, 87:3336-3343).

The receptor VEGFR-3 is widely expressed on endothelial cells duringearly embryonic development, but as embryogenesis proceeds, it becomesrestricted to venous endothelium and then to the lymphatic endothelium(Kaipainen et al., 1994, Cancer Res., 54:6571-6577; Kaipainen et al.,1995, Proc. Natl. Acad. Sci. USA, 92:3566-3570). VEGFR-3 continues to beexpressed on lymphatic endothelial cells in adults. This receptor isessential for vascular development during embryogenesis. Targetedinactivation of both copies of the VEGFR-3 gene in mice resulted indefective blood vessel formation characterized by abnormally organizedlarge vessels with defective lumens, leading to fluid accumulation inthe pericardial cavity and cardiovascular failure at post-coital day9.5. On the basis of these findings it has been proposed that VEGFR-3 isrequired for the maturation of primary vascular networks into largerblood vessels. However, the role of VEGFR-3 in the development of thelymphatic vasculature could not be studied in these mice because theembryos died before the lymphatic system emerged. Nevertheless it isassumed that VEGFR-3 plays a role in development of the lymphaticvasculature and lymphangiogenesis given its specific expression inlymphatic endothelial cells during embryogenesis and adult life. This issupported by the finding that ectopic expression of VEGF-C, a ligand forVEGFR-3, in the skin of transgenic mice, resulted in lymphaticendothelial cell proliferation and vessel enlargement in the dermis.Furthermore this suggests that VEGF-C may have a primary function inlymphatic endothelium, and a secondary function in angiogenesis andpermeability regulation which is shared with VEGF (Joukov et al., 1996,EMBO J., 15:290-298).

Some inhibitors of the VEGF/VEGF-receptor system have been shown toprevent tumor growth via an anti-angiogenic mechanism; see Kim et al.,1993, Nature, 362:841-844 and Saleh et al., 1996, Cancer Res.,56:393-401.

As mentioned above, the VEGF family of growth factors are members of thePDGF family. PDGF plays an important role in the growth and/or motilityof connective tissue cells, fibroblasts, myofibroblasts and glial cells(Heldin et al., “Structure of platelet-derived growth factor:Implications for functional properties”, 1993, Growth Factor,8:245-252). In adults, PDGF stimulates wound healing (Robson et al.,1992, Lancet, 339:23-25). Structurally, PDGF isoforms aredisulfide-bonded dimers of homologous A- and B-polypeptide chains,arranged as homodimers (PDGF-AA and PDGF-BB) or a heterodimer (PDGF-AB).

PDGF isoforms exert their effects on target cells by binding to twostructurally related receptor tyrosine kinases (RTKs). Thealpha-receptor binds both the A- and B-chains of PDGF, whereas thebeta-receptor binds only the B-chain. These two receptors are expressedby many cell lines grown in vitro, and are mainly expressed bymesenchymal cells in vivo. The PDGFs regulate cell proliferation, cellsurvival and chemotaxis of many cell types in vitro (reviewed in Heldinet al., 1998, Biochim Biophys Acta., 1378:F79-113). In vivo, they exerttheir effects in a paracrine mode since they often are expressed inepithelial (PDGF-A) or endothelial cells (PDGF-B) in close apposition tothe PDGFR expressing mesenchyme. In tumor cells and in cell lines grownin vitro, coexpression of the PDGFs and the receptors generate autocrineloops which are important for cellular transformation (Betsholtz et al.,1984, Cell, 39:447-57; Keating et al., 1990, J. R. Coll Surg Edinb.,35:172-4). Overexpression of the PDGFs have been observed in severalpathological conditions, including malignancies, arteriosclerosis, andfibroproliferative diseases (reviewed in Heldin et al., 1996, TheMolecular and Cellular Biology of Wound Repair, New York: Plenum Press,249-273).

The importance of the PDGFs as regulators of cell proliferation andsurvival are well illustrated by recent gene targeting studies in micethat have shown distinct physiological roles for the PDGFs and theirreceptors despite the overlapping ligand specificities of the PDGFRs.Homozygous null mutations for either of the two PDGF ligands or thereceptors are lethal. Approximately 50% of the homozygous PDGF-Adeficient mice have an early lethal phenotype, while the survivinganimals have a complex postnatal phenotype with lung emphysema due toimproper alveolar septum formation because of a lack of alveolarmyofibroblasts (Boström et al., 1996, Cell, 85:863-873). The PDGF-Adeficient mice also have a dermal phenotype characterized by thindermis, misshapen hair follicles and thin hair (Karlsson et al., 1999,Development, 126:2611-2). PDGF-A is also required for normal developmentof oligodendrocytes and subsequent myelination of the central nervoussystem (Fruttiger et al., 1999, Development, 126:457-67). The phenotypeof PDGFR-alpha deficient mice is more severe with early embryonic deathat E10, incomplete cephalic closure, impaired neural crest development,cardiovascular defects, skeletal defects, and edemas (Soriano et al.,1997, Development, 124:2691-70). The PDGF-B and PDGFR-beta deficientmice develop similar phenotypes that are characterized by renal,hematological and cardiovascular abnormalities (Levéen et al., 1994,Genes Dev., 8:1875-1887; Soriano et al., 1994, Genes Dev., 8:1888-96;Lindahl et al., 1997, Science, 277:242-5; Lindahl, 1998, Development,125:3313-2), where the renal and cardiovascular defects, at least inpart, are due to the lack of proper recruitment of mural cells (vascularsmooth muscle cells, pericytes or mesangial cells) to blood vessels(Levéen et al., 1994, Genes Dev., 8:1875-1887; Lindahl et al., 1997,Science, 277:242-5; Lindahl et al., 1998, Development, 125:3313-2).

Most recently, an additional member of the PDGF/VEGF family of growthfactors was identified, PDGF-C. PDGF-C is described in InternationalPatent Application PCT/US99/22668 (WO 00/18212), filed Sep. 30, 1999.PDGF-C has a two-domain structure not previously recognized within thisfamily of growth factors, a N-terminal C1r/C1s/embryonic sea urchinprotein Uegf/bone morphogenetic protein 1 (CUB) domain, and a C-terminalPDGF/VEGF homology domain (P/VHD). The structure of the P/VHD in PDGF-Cshows a low overall sequence identity with other PDGF/VEGF homologydomains, although the eight invariant cysteine residues involved ininter- and intra-molecular disulfide bond formation are present. Thecysteine spacing in the central, most conserved region of this domain isdifferent from other PDGF/VEGF domains, with an insertion of three aminoacid residues. Despite the fact that the insertion occurs close to theloop 2 region which has been proposed to be involved in receptorbinding, it was shown that this domain of PDGF-CC binds PDGFR-alpha withalmost identical affinities as homodimers of PDGF-A or -B chains. Inaddition, four extra cysteine residues are present in this domain. Fulllength and truncated PDGF-CC were found not to bind to VEGFR-1, -2 or-3, or to PDGFR-beta.

PDGF-C requires proteolytic removal of the N-terminal CUB domain forreceptor binding and activation of the receptor. This indicates that theCUB domains are likely to sterically block the receptor binding epitopesof the unprocessed dimer. The in vitro and in vivo proteolyticallyprocessed proteins are devoid of N-terminal portions corresponding tomore than 14-16 kDa as determined from SDS-PAGE analysis which isconsistent with a loss of the 110 amino acid long CUB domain and a partof the hinge region between the CUB and core domains that vary inlength.

PDGF-C is not proteolytically processed during secretion in transfectedCOS cells indicating that proteolytic removal of the CUB domain occursextracellularly, and not during secretion. This is in contrast to PDGF-Aand -B (Östman et al., 1992, J. Cell. Biol., 118:509-519) which appearto be processed intracellularly by furin-like endoproteases (Nakayama etal., 1997, Biochem J., 327:625-635).

Northern blots show PDGF-C mRNA in a variety of human tissues, includingheart, liver, kidney, pancreas and ovary.

In situ localization studies demonstrate expression of PDGF-C in certainepithelial structures, and PDGFR-alpha in adjacent mesenchyme,indicating the potential of paracrine signaling in the developingembryo. PDGF-C expression seems particularly abundant at sites ofongoing ductal morphogenesis, indicating a role of the factor inconnective tissue remodeling at these sites. The expression pattern isdistinct from that of PDGF-A or PDGF-B indicating that the three growthfactors have different roles despite their similar PDGFR-alpha bindingand signaling activities. This is illustrated by the mouse embryonickidney, in which PDGF-C is expressed in early aggregates of metanephricmesenchyme undergoing epithelial conversion, whereas PDGF-A is expressedin more mature tubular structures, and PDGF-B by vascular endothelialcells. PDGFR-alpha is expressed in the mesenchyme of the kidney cortex,adjacent to the sites of PDGF-C expression, indicating that thismesenchyme may be targeted specifically by PDGF-C. Indeed, PDGFR-alpha−/− mouse embryos show an extensive loss of the cortical mesenchymeadjacent to sites of PDGF-C expression, not seen in PDGF-A −/− mice orin PDGF-A/B −/− mice, indicating that PDGF-C has an essential role inthe development of kidney mesenchyme.

SUMMARY OF THE INVENTION

The invention generally provides an isolated novel growth factor,PDGF-D, a polypeptide that has the ability to stimulate, or enhance, orboth, one or more of proliferation, differentiation, growth, andmotility of cells expressing a PDGF-D receptor. The cells affected bythe inventive growth factor include, but are not limited to, endothelialcells, connective tissue cells, myofibroblasts and glial cells. Theinvention also provides isolated polynucleotide molecules encoding thenovel growth factor, and compositions useful for diagnostic and/ortherapeutic applications.

According to one aspect, the invention provides an isolated nucleic acidmolecule which comprises a polynucleotide sequence having at least 85%identity, more preferably at least 90%, and still more preferably atleast 95% identity, and most preferably at 100% identity to at leastnucleotides 1 to 600 of the sequence set out SEQ ID NO:3, at leastnucleotides 1 to 966 of the sequence set out in SEQ ID NO:5, at leastnucleotides 176 to 1285 of the sequence set out in SEQ ID NO:7, at leastnucleotides 935 to 1285 set out in SEQ ID NO:7, at least nucleotides 1to 1110 of SEQ ID NO:35, at least nucleotides 1-1092 of SEQ ID NO:37, orSEQ ID NO:39. The sequence of at least nucleotides 1 to 600 of thesequence set out in FIG. 3 (SEQ ID NO:3) or at least nucleotides 1 to966 of the sequence set out in FIG. 5 (SEQ ID NO:5) encodes a5′-truncated polypeptide, designated PDGF-D (formerly designated“VEGF-G”), while at least nucleotides 176 to 1285 of the sequence setout in FIG. 7 (SEQ ID NO:7) encodes a full-length PDGF-D. The sequenceof at least nucleotides 1 to 1110 of SEQ ID NO:35 encodes a murinePDGF-D, while the sequence of at least nucleotides 1-1092 of SEQ IDNO:37 encodes an identical protein as SEQ ID NO:35 except for a sixamino acid residue gap (a.a. #42-47) from the region between the signalsequence and the CUB domain (see below for details), and SEQ ID NO:39 aC-terminal truncated protein of the polypeptide encoded by SEQ ID NO:35.The PDGF-D polynucleotide of the invention can be a naked polynucleotideand/or in a vector or liposome.

PDGF-D is structurally homologous to PDGF-A, PDGF-B, VEGF, VEGF-B,VEGF-C and VEGF-D. The sequence of at least nucleotides 935 to 1285 setout in FIG. 7 (SEQ ID NO:7) encodes a portion of the PDGF/VEGF homologydomain, which is the bioactive fragment of PDGF-D. This bioactivefragment would also be encoded by the sequence of at least nucleotides 1to 600 of the sequence set out in FIG. 3 (SEQ ID NO:3) or at leastnucleotides 1 to 966 of the sequence set out in FIG. 5 (SEQ ID NO:5).

According to a second aspect, the PDGF-D polypeptide of the inventionhas the ability to stimulate and/or enhance proliferation and/ordifferentiation and/or growth and/or motility of cells expressing aPDGF-D receptor including, but not limited to, endothelial cells,connective tissue cells, myofibroblasts and glial cells and comprises asequence of amino acids having at least 85% identity, more preferably atleast 90%, and still more preferably at least 95% identity, and mostpreferably at 100% identity to the amino acid sequence set out in SEQ IDNOs:4, 6, 8, 36, 38 or 40, or a fragment or analog thereof which hasPDGF-D activity.

A preferred fragment is a truncated form of PDGF-D comprising a portionof the PDGF/VEGF homology domain (PVHD) of PDGF-D. The portion of thePVHD is from residues 254-370 of FIG. 8 (SEQ ID NO:8) where the putativeproteolytic processing site RKSK starts at amino acid residue 254 (SEQID NO:8). However, the PVHD extends toward the N terminus up to residue234 of FIG. 8 (SEQ ID NO:8). Herein the PVHD is defined as truncatedPDGF-D. The truncated PDGF-D is the putative activated form of PDGF-D.

Another preferred fragment is a truncated form of PDGF-D comprising onlythe CUB domain, as exemplified by the sequence set forth in SEQ IDNO:40. There may exist PDGF-D receptors, other than PDGFR-beta, thatbind to the unprocessed or un-cleaved factor (CUB+PDGF-homology domain).The CUB domain alone may bind to these receptors and would preventactivation of said receptors by blocking the receptors from binding toun-cleaved factors.

As used in this application, percent sequence identity is determined byusing the alignment tool of “MEGALIGN” from the Lasergene package(DNASTAR, Ltd. Abacus House, Manor Road, West Ealing, London W130ASUnited Kingdom). The MEGALIGN is based on the J. Hein method (Methods inEnzymology, 1990 183 626-645). The PAM 250 residue weight table is usedwith a gap penalty of eleven and a gap length penalty of three and aK-tuple value of two in the pairwise alignments. The alignment is thenrefined manually, and the number of identities are estimated in theregions available for a comparison.

Preferably the polypeptide has the ability to stimulate one or more ofproliferation, differentiation, motility, survival or vascularpermeability of cells expressing a PDGF-D receptor including, but notlimited to, vascular endothelial cells, lymphatic endothelial cells,connective tissue cells (such as fibroblasts), myofibroblasts and glialcells. Preferably the polypeptide has the ability to stimulate woundhealing. PDGF-D also has antagonistic effects on cells. For example, anantagonistic PDGF-D variant would be a partial PDGF-D moleculecontaining one intact full-length chain and one processed chain as adisulphide-linked dimer. In principle such a molecule would bemonovalent and bind to single PDGFR-beta receptors, but prevent theirdimerization thereby blocking signal transduction. These antagonisticactivities are also included in the biological activities of PDGF-D.Collectively, both the stimulating and antagonistic abilities arereferred to hereinafter as “biological activities of PDGF-D” and can bereadily tested by methods known in the art.

In another preferred aspect, the invention provides a polypeptidecomprising an amino acid sequence: PXCLLVXRCGGNCGC (SEQ ID NO:25)which is unique to PDGF-D and differs from the other members of thePDGF/VEGF family of growth factors because of the insertion of the threeamino acid residues (NCG) between the third and fourth cysteines (seeFIG. 9).

Polypeptides comprising conservative substitutions, insertions, ordeletions, but which still retain a biological activity of PDGF-D arewithin the scope of the invention. Persons skilled in the art will bewell aware of methods which can readily be used to generate suchpolypeptides, for example the use of site-directed mutagenesis, orspecific enzymatic cleavage and ligation. The skilled person will alsobe aware that peptidomimetic compounds or compounds in which one or moreamino acid residues are replaced by a non-naturally occurring amino acidor an amino acid analog may retain the required aspects of thebiological activity of PDGF-D. Such compounds can readily be made andtested for their ability to show the biological activity of PDGF-D byroutine activity assay procedures such as the fibroblast proliferationassay and are also within the scope of the invention.

In addition, possible variant forms of the PDGF-D polypeptide which mayresult from alternative splicing, as are known to occur with VEGF andVEGF-B, and naturally-occurring allelic variants of the nucleic acidsequence encoding PDGF-D are within the scope of the invention. Examplesof such a variant include the polypeptides set forth in SEQ ID NOs: 38and 40. Allelic variants are well known in the art, and representalternative forms or a nucleic acid sequence which comprisesubstitution, deletion or addition of one or more nucleotides, but whichdo not result in any substantial functional alteration of the encodedpolypeptide.

Such variant forms of PDGF-D can be prepared by targeting non-essentialregions of the PDGF-D polypeptide for modification. These non-essentialregions are expected to fall outside the strongly-conserved regionsindicated in FIG. 9 (SEQ ID NOs:8 and 32). In particular, the growthfactors of the PDGF family, including PDGF-D, are dimeric. PDGF-Ddiffers slightly from VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A andPDGF-B because it shows complete conservation of only seven of the eightcysteine residues in the PVHD (Olofsson et al., 1996, Proc. Natl. Acad.Sci. USA, 93:2576-2581; Joukov et al., 1996, EMBO J., 15:290-298). Thesecysteines are thought to be involved in intra- and inter-moleculardisulfide bonding. Loops 1, 2 and 3 of each subunit, which are formed byintra-molecular disulfide bonding, are involved in binding to thereceptors for the PDGF/VEGF family of growth factors (Andersson et al.,1995, Growth Factors, 12:159-164).

Persons ordinarily skilled in the art thus are well aware that thesecysteine residues generally should be preserved and that the activesites present in loops 1, 2 and 3 also should be preserved. However,other regions of the molecule can be expected to be of lesser importancefor biological function, and therefore offer suitable targets formodification. Modified polypeptides can readily be tested for theirability to show the biological activity of PDGF-D by routine activityassay procedures such as the fibroblast proliferation assay.

It is contemplated that some modified PDGF-D polypeptides will have theability to bind to PDGF-D receptors on cells including, but not limitedto, endothelial cells, connective tissue cells, myofibroblasts and/orglial cells, but will be unable to stimulate cell proliferation,differentiation, migration, motility or survival or to induce vascularproliferation, connective tissue development or wound healing. Thesemodified polypeptides are expected to be able to act as competitive ornon-competitive inhibitors of the PDGF-D polypeptides and growth factorsof the PDGF/VEGF family, and to be useful in situations where preventionor reduction of the PDGF-D polypeptide or PDGF/VEGF family growth factoraction is desirable. Thus such receptor-binding but non-mitogenic,non-differentiation inducing, non-migration inducing, non-motilityinducing, non-survival promoting, non-connective tissue promoting,non-wound healing or non-vascular proliferation inducing variants of thePDGF-D polypeptide are also within the scope of the invention, and arereferred to herein as “receptor-binding but otherwise inactivevariants.” Because PDGF-D forms a dimer in order to activate its onlyknown receptor, it is contemplated that one monomer comprises thereceptor-binding but otherwise inactive variant modified PDGF-Dpolypeptide and a second monomer comprises a wild-type PDGF-D or awild-type growth factor of the PDGF/VEGF family. These dimers can bindto its corresponding receptor but cannot induce downstream signaling.

It is also contemplated that there are other modified PDGF-Dpolypeptides that can prevent binding of a wild-type PDGF-D or awild-type growth factor of the PDGF/VEGF family to its correspondingreceptor on cells including, but not limited to, endothelial cells,connective tissue cells (such as fibroblasts), myofibroblasts and/orglial cells. Thus these dimers will be unable to stimulate endothelialcell proliferation, differentiation, migration, survival, or inducevascular permeability, and/or stimulate proliferation and/ordifferentiation and/or motility of connective tissue cells,myofibroblasts or glial cells. These modified polypeptides are expectedto be able to act as competitive or non-competitive inhibitors of thePDGF-D growth factor or a growth factor of the PDGF/VEGF family, and tobe useful in situations where prevention or reduction of the PDGF-Dgrowth factor or PDGF/VEGF family growth factor action is desirable.Such situations include the tissue remodeling that takes place duringinvasion of tumor cells into a normal cell population by primary ormetastatic tumor formation. Thus such PDGF-D or PDGF/VEGF family growthfactor-binding but non-mitogenic, non-differentiation inducing,non-migration inducing, non-motility inducing, non-survival promoting,non-connective tissue promoting, non-wound healing or non-vascularproliferation inducing variants of the PDGF-D growth factor are alsowithin the scope of the invention, and are referred to herein as “thePDGF-D growth factor-dimer forming but otherwise inactive or interferingvariants.”

An example of a PDGF-D growth factor-dimer forming but otherwiseinactive or interfering variant is where the PDGF-D has a mutation whichprevents cleavage of CUB domain from the protein. It is furthercontemplated that a PDGF-D growth factor-dimer forming but otherwiseinactive or interfering variant could be made to comprise a monomer,preferably a monomer whose own N-terminal CUB domain has been removed(hereinafter a “CUB-removed monomer”) of VEGF, VEGF-B, VEGF-C, VEGF-D,PDGF-A, PDGF-B, PDGF-C, PDGF-D or PlGF linked to a CUB domain that has amutation which prevents cleavage of CUB domain from the protein. Dimersformed with the above mentioned PDGF-D growth factor-dimer forming butotherwise inactive or interfering variants and the monomers linked tothe mutant CUB domain would be unable to bind to their correspondingreceptors.

A variation on this contemplation would be to insert a proteolytic sitebetween a CUB-removed monomer of VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-A,PDGF-B, PDGF-C, PDGF-D or PlGF and the mutant CUB domain which isdimerized to a CUB-removed monomer of VEGF, VEGF-B, VEGF-C, VEGF-D,PDGF-A, PDGF-B, PDGF-C, PDGF-D or PlGF. Addition of the specificprotease(s) for this proteolytic site would cleave the CUB domain andthereby release an activated dimer that can then bind to itscorresponding receptor. In this way, a controlled release of anactivated dimer is made possible.

According to a third aspect, the invention provides a purified andisolated nucleic acid encoding a polypeptide or polypeptide fragment ofthe invention as defined above. The nucleic acid may be DNA, genomicDNA, cDNA or RNA, and may be single-stranded or double stranded. Thenucleic acid may be isolated from a cell or tissue source, or ofrecombinant or synthetic origin. Because of the degeneracy of thegenetic code, the person skilled in the art will appreciate that manysuch coding sequences are possible, where each sequence encodes theamino acid sequence shown in FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:6)or FIG. 8 (SEQ ID NO:8), a bioactive fragment or analog thereof, areceptor-binding but otherwise inactive or partially inactive variantthereof or a PDGF-D dimer-forming but otherwise inactive or interferingvariants thereof.

A fourth aspect of the invention provides vectors comprising the cDNA ofthe invention or a nucleic acid molecule according to the third aspectof the invention, and host cells transformed or transfected with nucleicacids molecules or vectors of the invention. These may be eukaryotic orprokaryotic in origin. These cells are particularly suitable forexpression of the polypeptide of the invention, and include insect cellssuch as Sf9 cells, obtainable from the American Type Culture Collection(ATCC SRL-171), transformed with a baculovirus vector, and the humanembryo kidney cell line 293-EBNA transfected by a suitable expressionplasmid. Preferred vectors of the invention are expression vectors inwhich a nucleic acid according to the invention is operatively connectedto one or more appropriate promoters and/or other control sequences,such that appropriate host cells transformed or transfected with thevectors are capable of expressing the polypeptide of the invention.Other preferred vectors are those suitable for transfection of mammaliancells, or for gene therapy, such as adenoviral-, vaccinia- orretroviral-based vectors or liposomes. A variety of such vectors isknown in the art.

The invention also provides a method of making a vector capable ofexpressing a polypeptide encoded by a nucleic acid molecule according tothe invention, comprising the steps of operatively connecting thenucleic acid molecule to one or more appropriate promoters and/or othercontrol sequences, as described above.

The invention further provides a method of making a polypeptideaccording to the invention, comprising the steps of expressing a nucleicacid or vector of the invention in a host cell, and isolating thepolypeptide from the host cell or from the host cell's growth medium.

In yet a further aspect, the invention provides an antibody specificallyreactive with a polypeptide of the invention or a fragment of thepolypeptide. This aspect of the invention includes antibodies specificfor the variant forms, immunoreactive fragments, analogs andrecombinants of PDGF-D. Such antibodies are useful as inhibitors orantagonists of PDGF-D and as diagnostic agents for detecting andquantifying PDGF-D. Polyclonal or monoclonal antibodies may be used.Monoclonal and polyclonal antibodies can be raised against polypeptidesof the invention or fragment or analog thereof using standard methods inthe art. In addition the polypeptide can be linked to an epitope tag,such as the FLAG® octapeptide (Sigma, St. Louis, Mo.), to assist inaffinity purification. For some purposes, for example where a monoclonalantibody is to be used to inhibit effects of PDGF-D in a clinicalsituation, it may be desirable to use humanized or chimeric monoclonalantibodies. Such antibodies may be further modified by addition ofcytotoxic or cytostatic drug(s). Methods for producing these, includingrecombinant DNA methods, are also well known in the art.

This aspect of the invention also includes an antibody which recognizesPDGF-D and is suitably labeled.

Polypeptides or antibodies according to the invention may be labeledwith a detectable label, and utilized for diagnostic purposes.Similarly, the thus-labeled polypeptide of the invention may be used toidentify its corresponding receptor in situ. The polypeptide or antibodymay be covalently or non-covalently coupled to a suitable supermagnetic,paramagnetic, electron dense, ecogenic or radioactive agent for imaging.For use in diagnostic assays, radioactive or non-radioactive labels maybe used. Examples of radioactive labels include a radioactive atom orgroup, such as ¹²⁵I or ³²P. Examples of non-radioactive labels includeenzymatic labels, such as horseradish peroxidase or fluorimetric labels,such as fluorescein-5-isothiocyanate (FITC). Labeling may be direct orindirect, covalent or non-covalent.

Clinical applications of the invention include diagnostic applications,acceleration of angiogenesis in tissue or organ transplantation topromote graft growth and vascularization, or stimulation of woundhealing, or connective tissue development, or to establish collateralcirculation in tissue infarction or arterial stenosis, such as coronaryartery disease, and inhibition of angiogenesis in the treatment ofcancer or of diabetic retinopathy and inhibition of tissue remodelingthat takes place during invasion of tumor cells into a normal cellpopulation by primary or metastatic tumor formation. Quantitation ofPDGF-D in cancer biopsy specimens may be useful as an indicator offuture metastatic risk.

PDGF-D may also be relevant to a variety of lung conditions. PDGF-Dassays could be used in the diagnosis of various lung disorders. PDGF-Dcould also be used in the treatment of lung disorders to improve bloodcirculation in the lung and/or gaseous exchange between the lungs andthe blood stream. Similarly, PDGF-D could be used to improve bloodcirculation to the heart and O₂ gas permeability in cases of cardiacinsufficiency. In a like manner, PDGF-D could be used to improve bloodflow and gaseous exchange in chronic obstructive airway diseases.

Thus the invention provides a method for stimulating angiogenesis,lymphangiogenesis, neovascularization, connective tissue developmentand/or wound healing in a mammal in need of such treatment, comprisingthe step of administering an effective dose of PDGF-D, or a fragment oran analog thereof which has the biological activity of PDGF-D to themammal. The PDGF-D polypeptides may be administered either in the formof its bioactive fragment (e.g. residues 254-370 of SEQ ID NO: 8), or inthe form of a full-length sequence which may be activated, e.g. with asuitable protease, in situ. Alternatively, a nucleic acid moleculecoding for a bioactive PDGF-D polypeptide may be administered, or anucleic acid molecule coding for a full-length PDGF-D polypeptidetogether with a nucleic acid molecule coding for a suitable protease areadministered together, preferably under the control of regulatoryelements suitable for regulation of their respective expression.Optionally the PDGF-D, or fragment or analog thereof may be administeredtogether with, or in conjunction with, one or more of VEGF, VEGF-B,VEGF-C, VEGF-D, PlGF, PDGF-A, PDGF-B, PDGF-C, FGF and/or heparin.

PDGF-D polypeptides may be directly delivered to the site of interestwhere angiogenesis etc are desired. Numerous direct polypeptide deliverymethods are known and may be used. See e.g. Talmadge, 1993, Thepharmaceutics and delivery of therapeutic polypeptides and proteins,Adv. Drug Del. Rev. 10:247-299. The polypeptides may be administeredorally. Although polypeptides are generally known to have pooravailability through oral administration, various methods known in theart have been developed to overcome this limitation. For example,biodegradable polymeric matrices have been used for delivering proteinsover a desired period of time. For example, the use of biodegradablepoly(d,l-lactic-co-glycolic acid) (PLGA) microspheres for the deliveryof peptides and proteins has been widely reported (Mehta et al., 1996,Peptide containing microspheres from low molecular weight andhydrophilic poly(d,l-lactide-co-glycolide), J. Control Release41:249-257; Chiba et al., 1997, Controlled protein delivery frombiodegradable tyrosine-containing poly(anhydride-co-imide) microspheres.Biomaterials 18:893-901; Ravivarapu et al., 2000, Polymer andmicrosphere blending to alter the release of a peptide from PLGAmicrospheres, Eur. J. Pharm. Biopharm. 50:263-270).

Preferably, direct application of the polypeptides, especially directinjection, may be used. Because wound-healing and other conditionsrequiring enhanced angiogenesis typically require local application ofPDGF-D polypeptides and other growth factors for only a limited time,direct injection, even frequent direct injection of the polypeptides tothe desired site(s) is acceptable and is not likely to be very tediousor expensive and pose problems such as poor patient acceptance. Methodsof direct application of polypeptides are well-known to those ordinarilyskilled in the art, and recent successes, strategies, and potentials oftopical application of PDGF-BB in improving healing were reviewed byCupp et al., 2002, Gene therapy, electroporation, and the future ofwound-healing therapies, Facial Plast. Surg. 18:53-57.

In another preferred embodiment, the therapeutic polypeptides of thepresent invention may be delivered in the form of nucleic acid moleculesencoding the polypeptides. Many established and well-known methods forgene delivery or gene therapy may be used for administering genes orother nucleic acid molecules encoding PDGF-D to the patient. See e.g.Rubany, 2001, The future of human gene therapy, Mol. Aspects. Med.22:113-42. A single dose of naked DNA of VEGF and PDGF was used to treatrats with cysteanmine-induced duodenal ulcers, and was shown tosignificantly accelerate chronic duodenal ulcer healing, and increaseVEGF and PDGF levels in duodenal mucosa (Szabo et al., 2001, GeneExpression and gene therapy in experimental duodena ulceration, J.Physiol. Paris 95:325-335).

The polynucleotides encoding PDGF-D preferably are linked operativelyunder the control of suitable promoter so that they are expressed whentaken up by the host cells. PDGF-D is a diffusible protein, and as suchit will exert its effects on cells directly expressing the polypeptides,as well as on surrounding cells. Accordingly, suitable promoters may beconstitutive promoters such as promoter and enhancer elements fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), and SV40, and the ratbeta-actin promoter. Preferably, inducible or tissue specific promotersare used to increase expression level, improve specificity and reduceside effects. In this regard, suitable promoters include the keratin 5(K5) promoter (Pierce et al., 1998, Oncogene 16: 1267-1276; Pierce etal., 1998, Proc. Natl. Acad. Sci. USA 95:8858-8863), the Cyr61 promoter(inducible in granulation tissue during wound healing) (Latinkic et al.,2001, Promoter function of the angiogenic inducer Cyr61 gene intransgenic mice: tissue specificity, inducibility during wound healing,and role of the serum response element, Endocrinol. 142:2549-2557), andthe FAP promoter (Neidermeyer et al., 2001, Expression of the fibroblastactivation protein during mouse embryo development, Int. J. Dev. Biol.45:445-447).

Suitable polynucleotides may also be delivered as nonviral vectors,using methods well-known to those ordinarily skilled in the art. Seee.g. Brown et al., 2001, Gene delivery with synthetic (non-viral)carriers, Int. J. Pharm. 229:1-21; and Pouton et al., 1998, Key issuesin non-viral gene delivery, Adv. Drug Deliv. Rev. 34:3-19.).Lipofection, liposome mediated gene transfer are preferred (Romano etal., 1999, Gene transfer technology in therapy: current applications andfuture goals. Stem Cells 17:191-202; Mountain, 2000, Gene therapy: thefirst decade. Trends. Biotechnol. 18:119-28; Mhashilkar et al., 2001,Gene therapy: Therapeutic approaches and implications. Biotechnol. Adv.19:279-97; and Lasic, 1998, Novel applications of liposomes, TrendsBiotechnol. 16:307-21).

One of the simplest ideas for non-viral gene delivery is the use ofpurified DNA in the form of plasmids. A naked polynucleotide operativelycoding for the polypeptide may be delivered, along with apharmaceutically acceptable carrier, directly to the desired site, wherethe polynucleotide is taken up by the cells at the site and expressed orotherwise exerts its therapeutic effects. This is particularly preferredif transient expression of the gene is desired. The transfer of nakedDNA by physical means is well known, by such means as gene guns andelectroporation. See e.g. Spack et al., 2001, Developing non-viral DNAdelivery systems for cancer and infectious disease, DDT 6:186-97. Seealso Cupp et al., 2002, supra.

In general, RNA molecules will have more transient effects than DNAmolecules. The effects of the naked RNA molecules so delivered lasttypically for less than about 20 days, usually less than 10 days, andoften less than 3 to 5 days. Delivery may be by injection, spray,biolistic methods, and so on, depending on the site.

In another embodiment, suitable polynucleotides may also be deliveredwithin viral vectors, which are known to have higher transfectionefficiency compared to nonviral vectors. See e.g. Robbins et al., 1998,Viral vectors for gene therapy, Pharmacol. Ther. 80:35-47; and Kay etal., 2001, Viral vectors for gene therapy: the art of turning infectiousagents into vehicles of therapeutics, Nat. Med. 7:33-40. Suitable viralvectors include those derived from retroviruses (including lentivirues)(see e.g. Breithart et al., 1999, Ann. Plast. Surg. 43:632-9),especially the Moloney murineleukemia virus and pseudotyped retroviruses(Chen et al., 2001, Safety testing for replication-competent retrovirusassociated with gibbon apeleukemia virus-pseudotyped retroviral vectors.Hum. Gene. Ther. 12:61-70); adenoviruses, especially the thirdgeneration “gutless” adenoviral vector (Kochanek et al., 2001,High-capacity “gutless” adenoviral vectors. Curr. Opin. Mol. Ther.3:454-63.); chimeric viruses that combine the advantages of bothretroviruses and adenoviruses (Reynolds et al., 1999, Chimeric viralvectors-the best of both worlds? Mol. Med. Today 5:25-31, 1999);adeno-associated virus (Ponnazhagan et al., 2001, Adeno-associated virusfor gene therapy. Cancer Res., 61:6313-21; and Monahan et al., 2000,Adeno-associated virus vectors for gene therapy: more pros than cons?Mol. Med. Today, 6:433-40.); vaccinia viruses (Peplinski, et al., 1998,Vaccinia virus for human gene therapy. Surg. Oncol. Clin. N. Am.,7:575-588); and herpes simplex virus (Latchman. 2001, Gene delivery andgene therapy with herpes simplex virus-based vectors. Gene 264:1-9).

Adenoviral vectors are preferred. Chen et al. (2002) showed thatrecombinant adenoviruses encoding the PDGF-A gene express and secretePDGF-A in vitro, and induce sustained down regulation of PDGFαR encodedby the growth arrest specific (gas) gene (Am. J. Physiol. Cell Physiol.282:C538-44). Szabo et al., supra, used a single dose of adenoviralvectors expressing VEGF and PDGF to treat rats with cysteanmine-inducedduodenal ulcers, and showed significant acceleration of chronic duodenalulcer healing, and increased VEGF and PDGF levels in duodenal mucosa.Giannobile et al., 2001, J. Periodontol. 72:815-23 showed thatadenoviral vectors expressing PDGF-A stimulated cementoblast DNAsynthesis and subsequent proliferation. Zhu et al., 2001, J. Dent. Res.80:892-7 demonstrated that adenoviruses encoding PDGF-A enhancedmitogenic and proliferative responses in osteoblasts, periodontalligament fibroblasts and gingival fibroblasts. See also Liechty et al.,1999, Adenoviral mediated overexpression of PDGF-B corrects ischemicimpaired wound healing, J. Invest. Dermatol. 113:375-83.

The effects of vectors coding for PDGF-D polypeptides may also beimproved with matrix immobilization to enhance tissue repair activity.Biocompatible matrices capable of immobilizing adenoviral vectors havebeen successfully used in treating ischemic excisional wounds.Specifically, collagen-formulated vectors encoding PDGF-B, whendelivered as subcutaneously implanted sponges, have been shown toenhance granulation tissue deposition, enhance epithelial area, andimprove wound closure more effectively than aqueous formulations of thesame vectors. With longer time, complete healing without excessive scarformation was achieved. In comparison, aqueous formulations allowedvector seepage and led to PDGF-induced hyperplasia in surroundingtissues but not in wound beds. In addition, repeated applications ofPDGF-BB proteins were required for neotissue induction approachingequivalence to a single application of collagen-immobilized vectors.(Doukas et al., 2002, Hum. Gene Ther. 12:783-98). In the same study,Doukas et al. also showed that vectors encoding fibroblast growth factor2 or vascular endothelial growth factor promoted primarily angiogenicresponses. Similar improvements were observed in dermal ulcer wounds inthe ears of young adult New Zealand white rabbits with collagen embeddedPDGF-B or PDGF-A DNA plasmids (Tyrone et al., 2000, J. Surg. Res.93:230-6); in soft tissue repair by enhancing de novo tissue formation(Chandler et al., 2000, Mol. Ther. 2:153-60).

Other materials may also be used as sustained release matrices fordelivering vectors encoding PDGF genes. For example, matrices ofpoly(lactide-co-glycolide) (PLG) were loaded with plasmids and shown torelease the plasmids over a period ranging from days to months in vitro,and led to the transfection of large numbers of cells. In vivo deliveryenhanced matrix deposition and blood vessel formation in the developingtissue (Shea et al., 1999, Nat. Biotechnol. 17:551-4).

Another method of gene delivery uses fusigenic virosomes. This approachcombines some of the advantages of viral delivery vectors with thesafety and ‘simplicity’ of the liposome to produce fusigenic virosomes(Dzau et al., 1996, Fusigenic viral liposome for gene therapy incardiovascular diseases. Proc Natl. Acad. Sci. USA 93:11421-25).Virosomes have been engineered by complexing the membrane fusionproteins of haemagglutinating virus of Japan (HVJ, which is also knownas Sendai virus) with either liposomes that already encapsulate plasmidDNA or oligodeoxynucleotides (ODN) for antisense applications. Theinherent ability of the viral proteins in virosomes to cause fusion withcell membranes means that these hybrid vectors can be very efficient atintroducing their nucleic acid to the target cell, resulting in goodgene expression. Each viral vector has a limit on the size of transgenethat can be incorporated into its genome; no such limit exists forvirosome or liposome technology. Genes of up to 100 kilobase pairs havebeen delivered by fusigenic virosomes to cells both ex vivo and in vivo.

A further embodiment of the invention utilizes DNA-ligand conjugates fordelivery of genes encoding the PDGF-polypeptides. DNA-ligand conjugateshave two main components: a DNA-binding domain and a ligand forcell-surface receptors. The transgene can therefore be guidedspecifically to the target cell, where it is internalized viareceptor-mediated endocytosis. Once the DNA-ligand complex is in theendocytic pathway, the conjugate is likely to be destroyed when theendosome fuses with a lysosome. To avoid this, an adenovirus-deriveddomain may be incorporated into the cell-surface receptor part of theligand (Curiel et al, 1992, High-efficiency gene transfer mediated byadenovirus coupled to DNA-polylysine complexes, Hum. Gene Ther.3:147-154). The conjugates then have the same specificity asadenoviruses, binding to a wide host-cell range; they also possess anadenovirus characteristic that allows the conjugate to leave theendosome and enter the cytoplasm (by a process known as endosomolysis)before the endosome is destroyed by a lysosome.

According to another embodiment of the invention, suitable host cellsmay be transformed with polynucleotides, preferably vectors, morepreferably viral vectors, encoding the PDGF-D polypeptides of theinvention, and the host cells expressing the PDGF-D polypeptides may beintroduced to a host animal in need of wound healing or other treatment.Many methods of in vitro cell transformation are known and wellestablished in the art, including CaPO₄ transfection, which is achemical method that has been successfully used by molecular biologistsfor many years to introduce transgenes into cells in vitro with arelatively good efficiency (10%). Mathisen et at. showed thatautoreactive memory Th2 T cells can be genetically modified so that uponengagement of self antigen they produce regenerative growth factors suchas PDGF-A capable of mediating tissue repair during autoimmune disease(Mathisen et al., 1999, J. Autoimmun. 13:31-8

Conversely, PDGF-D antagonists (e.g. antibodies and/or competitive ornoncompetitive inhibitors of binding of PDGF-D in both dimer formationand receptor binding) could be used to treat conditions, such ascongestive heart failure, involving accumulation of fluid in, forexample, the lung resulting from increases in vascular permeability, byexerting an offsetting effect on vascular permeability in order tocounteract the fluid accumulation. Administrations of PDGF-D could beused to treat malabsorptive syndromes in the intestinal tract, liver orkidneys as a result of its blood circulation increasing and vascularpermeability increasing activities.

Thus, the invention provides a method of inhibiting angiogenesis,lymphangiogenesis, neovascularization, connective tissue developmentand/or wound healing in a mammal in need of such treatment, comprisingthe step of administering an effective amount of an antagonist of PDGF-Dto the mammal. The antagonist may be any agent that prevents the actionof PDGF-D, either by preventing the binding of PDGF-D to itscorresponding receptor on the target cell, or by preventing activationof the receptor, such as using receptor-binding but otherwise inactivePDGF-D variants. Suitable antagonists include, but are not limited to,antibodies directed against PDGF-D; competitive or non-competitiveinhibitors of binding of PDGF-D to the PDGF-D receptor(s), such as thereceptor-binding or PDGF-D dimer-forming but non-mitogenic PDGF-Dvariants referred to above; and anti-sense nucleotide sequences asdescribed below. For example, a truncated PDGFβ receptor was shown toinhibit thrombosis and neointima formation in an avian arterial injurymodel (Ding et al., 2001, Thromb. Haemost. 86:914-22).

In one embodiment, an antagonist of PDGF-D is a negative dominant mutantof a PDGF-D gene. This negative dominant mutant is able to inhibit theexpression of the native PDGF-D gene in the appropriate tissue of theanimal, thereby disrupting PDGF-D activity. See e.g. Chen et al., 2002,Am. J. Physiol. Cell Physiol. 282:C538-44 (showing that dominantnegative mutant of PDGF-A gene disrupts PDGF activity).

A method is provided for determining agents that bind to an activatedtruncated form of PDGF-D. The method comprises contacting an activatedtruncated form of PDGF-D with a test agent and monitoring binding by anysuitable means. Potential binding agents include proteins and othersubstances. The invention provides a screening system for discoveringagents that bind an activated truncated form of PDGF-D. The screeningsystem comprises preparing an activated truncated form of PDGF-D,exposing the activated truncated form of PDGF-D to a test agent, andquantifying the binding of said agent to the activated truncated form ofPDGF-D by any suitable means. The inhibitory effects of a binding agentare further determined by assaying the PDGF-D activities of the PDGF-Dpolypeptides bound with the binding agent. Both in vivo and in vitroassay methods may be used. Specifically, this screening system is usedto identify agents which inhibit the proteolytic cleavage of the fulllength PDGF-D protein and thereby prevent the release of the activatedtruncated form of PDGF-D. For this use, the full length PDGF-D isgenerally preferred.

Use of this screening system provides a means to determine compoundsthat may alter the biological function of PDGF-D. This screening methodmay be adapted to large-scale, automated procedures such as a PANDEX®(Baxter-Dade Diagnostics) system, allowing for efficient high-volumescreening of potential therapeutic agents.

For this screening system, an activated truncated form of PDGF-D or fulllength PDGF-D is prepared as described herein, preferably usingrecombinant DNA technology. A test agent, e.g. a compound or protein, isintroduced into a reaction vessel containing the activated truncatedform of or full length PDGF-D. Binding of the test agent to theactivated truncated form of or full length PDGF-D is determined by anysuitable means which include, but is not limited to, radioactively- orchemically-labeling the test agent. Binding of the activated truncatedform of or full length PDGF-D may also be carried out by a methoddisclosed in U.S. Pat. No. 5,585,277, which is incorporated byreference. In this method, binding of the test agent to the activatedtruncated form of or full length PDGF-D is assessed by monitoring theratio of folded protein to unfolded protein. Examples of this monitoringcan include, but are not limited to, monitoring the sensitivity of theactivated truncated form of or full length PDGF-D to a protease, oramenability to binding of the protein by a specific antibody against thefolded state of the protein.

Those of skill in the art will recognize that IC₅₀ values are dependenton the selectivity of the agent tested. For example, an agent with anIC₅₀ which is less than 10 nM is generally considered an excellentcandidate for drug therapy. However, an agent which has a loweraffinity, but is selective for a particular target, may be an evenbetter candidate. Those skilled in the art will recognize that anyinformation regarding the binding potential, inhibitory activity orselectivity of a particular agent is useful toward the development ofpharmaceutical products.

Where PDGF-D or a PDGF-D antagonist is to be used for therapeuticpurposes, the dose(s) and route of administration will depend upon thenature of the patient and condition to be treated, and will be at thediscretion of the attending physician or veterinarian. Suitable routesinclude oral, subcutaneous, intramuscular, intraperitoneal orintravenous injection, parenteral, topical application, implants etc.Topical application of PDGF-D may be used in a manner analogous to VEGF.Where used for wound healing or other use in which enhanced angiogenesisis advantageous, an effective amount of the truncated active form ofPDGF-D is administered to an organism in need thereof in a dose betweenabout 0.1 and 1000 μg/kg body weight.

The PDGF-D or a PDGF-D antagonist may be employed in combination with asuitable pharmaceutical carrier. The resulting compositions comprise atherapeutically effective amount of PDGF-D or a PDGF-D antagonist, and apharmaceutically acceptable non-toxic salt thereof, and apharmaceutically acceptable solid or liquid carrier or adjuvant.Examples of such a carrier or adjuvant include, but are not limited to,saline, buffered saline, Ringer's solution, mineral oil, talc, cornstarch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose,water, glycerol, ethanol, thickeners, stabilizers, suspending agents andcombinations thereof. Such compositions may be in the form of solutions,suspensions, tablets, capsules, creams, salves, elixirs, syrups, wafers,ointments or other conventional forms. The formulation should beconstituted to suit the mode of administration. Compositions whichcomprise PDGF-D may optionally further comprise one or more of PDGF-A,PDGF-B, PDGF-C, VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF and/or heparin.Compositions comprising PDGF-D will contain from about 0.1% to 90% byweight of the active compound(s), and most generally from about 10% to30%.

For intramuscular preparations, a sterile formulation, preferably asuitable soluble salt form of the truncated active form of PDGF-D, suchas hydrochloride salt, can be dissolved and administered in apharmaceutical diluent such as pyrogen-free water (distilled),physiological saline or 5% glucose solution. A suitable insoluble formof the compound may be prepared and administered as a suspension in anaqueous base or a pharmaceutically acceptable oil base, e.g. an ester ofa long chain fatty acid such as ethyl oleate.

According to yet a further aspect, the invention providesdiagnostic/prognostic devices typically in the form of test kits. Forexample, in one embodiment of the invention there is provided adiagnostic/prognostic test kit comprising an antibody to PDGF-D and ameans for detecting, and more preferably evaluating, binding between theantibody and PDGF-D. In one preferred embodiment of thediagnostic/prognostic device according to the invention, a secondantibody (the secondary antibody) directed against antibodies of thesame isotype and animal source of the antibody directed against PDGF-D(the primary antibody) is provided. The secondary antibody is coupleddirectly or indirectly to a detectable label, and then either anunlabeled primary antibody or PDGF-D is substrate-bound so that thePDGF-D/primary antibody interaction can be established by determiningthe amount of label bound to the substrate following binding between theprimary antibody and PDGF-D and the subsequent binding of the labeledsecondary antibody to the primary antibody. In a particularly preferredembodiment of the invention, the diagnostic/prognostic device may beprovided as a conventional enzyme-linked immunosorbent assay (ELISA)kit.

In another alternative embodiment, a diagnostic/prognostic device maycomprise polymerase chain reaction means for establishing sequencedifferences of a PDGF-D of a test individual and comparing this sequencestructure with that disclosed in this application in order to detect anyabnormalities, with a view to establishing whether any aberrations inPDGF-D expression are related to a given disease condition.

In addition, a diagnostic/prognostic device may comprise a restrictionlength polymorphism (RFLP) generating means utilizing restrictionenzymes and genomic DNA from a test individual to generate a pattern ofDNA bands on a gel and comparing this pattern with that disclosed inthis application in order to detect any abnormalities, with a view toestablishing whether any aberrations in PDGF-D expression are related toa given disease condition.

In accordance with a further aspect, the invention relates to a methodof detecting aberrations in PDGF-D gene structure in a test subjectwhich may be associated with a disease condition in the test subject.This method comprises providing a DNA sample from said test subject;contacting the DNA sample with a set of primers specific to PDGF-D DNAoperatively coupled to a polymerase and selectively amplifying PDGF-DDNA from the sample by polymerase chain reaction, and comparing thenucleotide sequence of the amplified PDGF-D DNA from the sample with thenucleotide sequences shown in FIG. 3 (SEQ ID NO:3), FIG. 5 (SEQ ID NO:5)or FIG. 7 (SEQ ID NO:7). The invention also includes the provision of atest kit comprising a pair of primers specific to PDGF-D DNA operativelycoupled to a polymerase, whereby said polymerase is enabled toselectively amplify PDGF-D DNA from a DNA sample.

The invention also provides a method of detecting PDGF-D in a biologicalsample, comprising the step of contacting the sample with a reagentcapable of binding PDGF-D, and detecting the binding. Preferably thereagent capable of binding PDGF-D is an antibody directed againstPDGF-D, particularly a monoclonal antibody. In a preferred embodimentthe binding and/or extent of binding is detected by means of adetectable label; suitable labels are discussed above.

In another aspect, the invention relates to a protein dimer comprisingthe PDGF-D polypeptide, particularly a disulfide-linked dimer. Theprotein dimers of the invention include both homodimers of PDGF-Dpolypeptide and heterodimers of PDGF-D and VEGF, VEGF-B, VEGF-C, VEGF-D,PlGF, PDGF-A, PDGF-B or PDGF-C.

According to a yet further aspect of the invention there is provided amethod for isolation of PDGF-D comprising the step of exposing a cellwhich expresses PDGF-D to heparin to facilitate release of PDGF-D fromthe cell, and purifying the thus-released PDGF-D.

Another aspect of the invention involves providing a vector comprisingan anti-sense nucleotide sequence which is complementary to at least apart of a DNA sequence which encodes PDGF-D or a fragment or analogthereof that has the biological activity of PDGF-D. In addition theanti-sense nucleotide sequence can be to the promoter region of thePDGF-D gene or other non-coding region of the gene which may be used toinhibit, or at least mitigate, PDGF-D expression.

According to a yet further aspect of the invention such a vectorcomprising an anti-sense sequence may be used to inhibit, or at leastmitigate, PDGF-D expression. The use of a vector of this type to inhibitPDGF-D expression is favored in instances where PDGF-D expression isassociated with a disease, for example where tumors produce PDGF-D inorder to provide for angiogenesis, or tissue remodeling that takes placeduring invasion of tumor cells into a normal cell population by primaryor metastatic tumor formation. Transformation of such tumor cells with avector containing an anti-sense nucleotide sequence would suppress orretard angiogenesis, and so would inhibit or retard growth of the tumoror tissue remodeling.

Another aspect of the invention relates to the discovery that the fulllength PDGF-D protein is likely to be a latent growth factor that needsto be activated by proteolytic processing to release an active PDGF/VEGFhomology domain. A putative proteolytic site is found in residues254-257 in the full length protein, residues —RKSK- (SEQ ID NO:9). Thisis a dibasic motif. The —RKSK- (SEQ ID NO:9) putative proteolytic siteis also found in PDGF-A, PDGF-B, VEGF-C and VEGF-D. In these fourproteins, the putative proteolytic site is also found just before theminimal domain for the PDGF/VEGF homology domain. Together these factsindicate that this is the proteolytic site.

Preferred proteases include, but are not limited, to plasmin, Factor Xand enterokinase. The N-terminal CUB domain may function as aninhibitory domain which might be used to keep PDGF-D in a latent form insome extracellular compartment and which is removed by limitedproteolysis when PDGF-D is needed.

According to this aspect of the invention, a method is provided forproducing an activated truncated form of PDGF-D or for regulatingreceptor-binding specificity of PDGF-D. These methods comprise the stepsof expressing an expression vector comprising a polynucleotide encodinga polypeptide having the biological activity of PDGF-D and supplying aproteolytic amount of at least one enzyme for processing the expressedpolypeptide to generate the activated truncated form of PDGF-D.

This aspect also includes a method for selectively activating apolypeptide having a growth factor activity. This method comprises thestep expressing an expression vector comprising a polynucleotideencoding a polypeptide having a growth factor activity, a CUB domain anda proteolytic site between the polypeptide and the CUB domain, andsupplying a proteolytic amount of at least one enzyme for processing theexpressed polypeptide to generate the activated polypeptide having agrowth factor activity.

In addition, this aspect includes the isolation of a nucleic acidmolecule which codes for a polypeptide having the biological activity ofPDGF-D and a polypeptide thereof which comprises a proteolytic sitehaving the amino acid sequence RKSK (SEQ ID NO:9) or a structurallyconserved amino acid sequence thereof.

Also this aspect includes an isolated dimer comprising an activatedmonomer of PDGF-D and an activated monomer of VEGF, VEGF-B, VEGF-C,VEGF-D, PDGF-D, PDGF-A, PDGF-B, PDGF-C or PlGF linked to a CUB domain,or alternatively, an activated monomer of VEGF, VEGF-B, VEGF-C, VEGF-D,PDGF-D, PDGF-A, PDGF-B or PlGF and an activated monomer of PDGF-D linkedto a CUB domain. The isolated dimer may or may not include a proteolyticsite between the activated monomer and the CUB domain.

Polynucleotides of the invention such as those described above,fragments of those polynucleotides, and variants of thosepolynucleotides with sufficient similarity to the non-coding strand ofthose polynucleotides to hybridize thereto under stringent conditionsall are useful for identifying, purifying, and isolating polynucleotidesencoding other, non-human, mammalian forms of PDGF-D. Thus, suchpolynucleotide fragments and variants are intended as aspects of theinvention. Exemplary stringent hybridization conditions are as follows:hybridization at 42° C. in 5×SSC, 20 mM NaPO₄, pH 6.8, 50% formamide;and washing at 42° C. in 0.2×SSC. Those skilled in the art understandthat it is desirable to vary these conditions empirically based on thelength and the GC nucleotide base content of the sequences to behybridized, and that formulas for determining such variation exist. Seefor example Sambrook et al, “Molecular Cloning: A Laboratory Manual”,Second Edition, pages 9.47-9.51, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory (1989).

Moreover, purified and isolated polynucleotides encoding other,non-human, mammalian PDGF-D forms also are aspects of the invention, asare the polypeptides encoded thereby and antibodies that arespecifically immunoreactive with the non-human PDGF-D variants. Thus,the invention includes a purified and isolated mammalian PDGF-Dpolypeptide and also a purified and isolated polynucleotide encodingsuch a polypeptide.

It will be clearly understood that nucleic acids and polypeptides of theinvention may be prepared by synthetic means or by recombinant means, ormay be purified from natural sources.

It will be clearly understood that for the purposes of thisspecification the word “comprising” means “included but not limited to.”The corresponding meaning applies to the word “comprises.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:1) shows a nucleotide sequence that includes a cDNAsequence encoding the C-terminal part of human PDGF-D (hPDGF-D). Thenucleotides which encode for the partial fragment of hPDGF-D are 1 to198. The deduced partial amino acid sequence of hPDGF-D (66 amino acidresidues-SEQ ID NO:2) derived from nucleotides 1 to 198 of FIG. 1 isshown in FIG. 2;

FIG. 3 (SEQ ID NO:3) shows an extended sequence of a partial human cDNAencoding for the hPDGF-D. The translated cDNA sequence is fromnucleotide 1 to 600. The deduced partial amino acid sequence of hPDGF-D(200 residues-SEQ ID NO:4) derived from nucleotides 1 to 600 of FIG. 3is shown in FIG. 4;

FIG. 5 shows a still further extended nucleotide sequence of a partialhuman cDNA. The nucleotides which encode for the 5′-truncatedfull-length hPDGF-D are 1 to 966 (SEQ ID NO:5). The deduced partialamino acid sequence of hPDGF-D (322 residues-SEQ ID NO:6) derived fromnucleotides 1 to 966 of FIG. 5 is shown in FIG. 6;

FIG. 7 (SEQ ID NO:7) shows the complete nucleotide sequence of cDNAencoding a hPDGF-D(1116 bp) and the deduced amino acid sequence offull-length hPDGF-D encoded thereby which consists of 370 amino acidresidues (FIG. 8-SEQ ID NO:8);

FIG. 9 shows an amino acid sequence alignment of the hPDGF-D withhPDGF-C (SEQ ID NOs:8 and 32, respectively);

FIG. 10 shows an amino acid sequence alignment of the PDGF/VEGF-homologydomain in hPDGF-D with several growth factors belonging to the VEGF/PDGFfamily (SEQ ID NOs:10-18, respectively);

FIG. 11 shows a phylogenetic tree of several growth factors belonging tothe VEGF/PDGF family;

FIG. 12 provides the amino acid sequence alignment of the CUB domainpresent in hPDGF-D (SEQ ID NO:19) and other CUB domains present in humanbone morphogenic protein-1 (hBMP-1, 3 CUB domains CUB1-3) (SEQ IDNOs:20-22, respectively) and in human neuropilin-1 (2 CUB domains) (SEQID NOs:23-24, respectively);

FIG. 13 shows the results of the SDS-PAGE analysis of human recombinantPDGF-D under reducing (R) and non-reducing (NR) conditions;

FIG. 14 shows the results of the immunoblot analysis of full-lengthPDGF-D and PDGF-C under reducing and non-reducing conditions employingaffinity-purified rabbit antibodies to full-length PDGF-D;

FIG. 15 provides that results of the relative expression levels ofPDGF-D (upper panel) and PDGF-B (lower panel) transcripts in severalhuman tissues as determined by Northern Blot analysis;

FIG. 16 shows PDGF-D expression in the developing kidney of a mouseembryo;

FIG. 17 shows a more detailed view of PDGF-D expression in thedeveloping kidney of a mouse embryo;

FIG. 18 shows a more detailed view of PDGF-D expression in thedeveloping kidney of a mouse embryo;

FIG. 19 shows that conditioned medium(CM)containing plasmin-digestedPDGF-D stimulates tyrosine phosphorylation of PDGFR-beta in PAE-1 cells;

FIG. 20 provides a graphical representation of the results of thecompetitive binding assay between plasmin-digested PDGF-D and PDGF-BBhomodimers for the PDGFRs-beta; and

FIG. 21 provides a graphical representation of the results of thecompetitive binding assay between plasmin-digested PDGF-D and PDGF-AAhomodimers for the PDGFRs-alpha.

FIG. 22A shows a schematic representation of the PDGF-D sequence of SEQID NO:35.

FIG. 22B shows a schematic representation of the PDGF-D sequence variantof SEQ ID NO:37, which corresponds to FIG. 22A but for 6 missing aminoacid residues.

FIG. 22C shows a schematic representation of the PDGF-D sequence variantof SEQ ID NO:39, which corresponds to FIG. 22A but for 6 missing aminoacid residues and the loss of a CUB domain in this sequence variant.

FIG. 23 shows a schematic representation of the PDGF-D sequence, notingthe spliced region from exon 5 to exon 7, removal of which yields thePDGF-D sequence variant of SEQ ID NO:39.

FIG. 24 shows SDS-PAGE analysis under reducing conditions of humanPDGF-DD formed from the core domain of factor Xa-digested mutantfull-length form of PDGF-D.

FIG. 25 shows the in vivo angiogenic activity of human PDGF-DD and otherPDGF isoforms in the mouse cornea pocket assay. In FIG. 25A-E, arrowspoint to where PDGF protein-containing beads were implanted.

FIG. 26 is a schematic diagram showing the K14-PDGF-D construct (SeeExample 11).

FIG. 27 shows a comparison of PDGF-D expression between K14-PDGF-Dtransgenic mouse (TG) and wild-type mouse (wt). Paraffin embedded mouseskin samples were stained with anti-PDGF-D. For experimental details,see Uutela et al., 2001, “Chromosomal location, exon structure andvascular expression patterns of the human PDGFC and PDGFD genes,”Circulation 103:2242-2247.

FIG. 28 shows a comparison of granulation tissue staining betweenK14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt) in wound areasafter two days. The samples were stained with the Van Gieson methodwhich stains elastin. The amount of granulation tissue is greater inPDGF-D positive mouse (TG).

FIG. 29 shows a comparison of granulation tissue staining betweenK14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt) in wound areasafter four days. The samples were stained with the Van Gieson methodwhich stains elastin. The amount of granulation tissue is greater inPDGF-D positive mouse (TG).

FIG. 30 shows a comparison of granulation tissue staining betweenK14-PDGF-D transgenic mouse (TG) and wild-type mouse (wt) in wound areasafter seven days. The samples were stained with the Van Gieson methodwhich stains elastin. The amount of granulation tissue is greater inPDGF-D positive mouse (TG).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows a nucleotide sequence of human cDNA which encodes aC-terminal portion of a novel growth factor, referred to herein asPDGF-D (formerly VEGF-G). PDGF-D is a new member of the VEGF/PDGFfamily. The nucleotide sequence of FIG. 1 (SEQ ID NO:1) was derived froma human EST sequence (id. AI488780) in the dbEST database at the NCBI inWashington, D.C. The nucleotides 1 to 198 of the cDNA of FIG. 1 (SEQ IDNO:1) encodes a 66 amino acid polypeptide (FIG. 2-SEQ ID NO:2) whichshows some sequence similarity to the known members of the VEGF/PDGFfamily.

The amino acid sequence of the polypeptide encoded by the nucleotides 1to 198 of the polynucleotide of FIG. 1 (SEQ ID NO:1) is shown in FIG. 2(SEQ ID NO:2).

To generate more sequence information on human PDGF-D, a human fetallung λgt10 cDNA library was screened using a 327 bp polymerase chainreaction (PCR)-generated probe, based on the originally identified ESTsequence. The probe was generated from DNA from a commercially availablehuman fetal lung cDNA library (Clontech) which was amplified by PCRusing two primers derived from the identified EST (AI488780). Theprimers were: 5′-GTCGTGGAACTGTCAACTGG (forward) (SEQ ID NO:26) and5′-CTCAGCAACCACTTGTGTTC (reverse). (SEQ ID NO:27)The amplified 327 bp fragment was cloned into the pCR2.1 vector(Invitrogen). Nucleotide sequencing verified that the insertcorresponded to the EST. The screen identified several positive clones.The inserts from two of these clones, clones 5 and 8 were subcloned intopBluescript and subjected to nucleotide sequencing using internal orvector-specific primers. The nucleotide sequences determined wereidentical in both clones and are shown in FIG. 3 (SEQ ID NO:3). Thecoding region of the 690 bp polynucleotide is nucleotides 1-600 (SEQ IDNO:3) that encodes for a large portion of hPDGF-D with the exception ofthe 5′-end. This portion of hPDGF-D includes the bioactive fragment ofhPDGF-D. The deduced partial amino acid sequence of hPDGF-D (200residues-SEQ ID NO:4) derived from nucleotides 1 to 600 of FIG. 3 (SEQID NO:3) is shown in FIG. 4 (SEQ ID NO:4).

Extended nucleotide sequencing of the isolated human PDGF-D cDNA clonesfrom this human fetal lung cDNA library has provided additionalsequence. FIG. 5 (SEQ ID NO:5) shows a nucleotide sequence of a partialhuman cDNA (1934 bp) that encodes hPDGF-D. The coding region of the 1934bp polynucleotide is nucleotides 1 to 966 that encodes for hPDGF-Dexcept for the most 5′-end of the polypeptide. The deduced partial aminoacid sequence of hPDGF-D (322 residues-SEQ ID NO:6) derived fromnucleotides 1 to 966 of FIG. 5 (SEQ ID NO:5) is shown in FIG. 6 (SEQ IDNO:6).

FIG. 7 (SEQ ID NO:7) shows a polynucleotide sequence of cDNA encoding afull-length hPDGF-D. The region encoding PDGF-D is 1116 bp. The deducedamino acid sequence of full-length hPDGF-D is 370 amino acid residues(FIG. 8-SEQ ID NO:8).

The sequence for the 5′ end of full-length PDGF-D was obtained usingRapid Amplification of cDNA Ends (RACE) PCR, and clones containing cDNAfrom the human heart (Marathon-Ready™ cDNA, Clontech, Cat# 7404-1).These cDNA clones have an adaptor sequence attached to the 5′ end ofeach clone, including a site for primer called Adaptor Primer 1(Clontech): 5′ CCATCCTAATACGACTCACTATAGGGC 3′. (SEQ ID NO:28)

This primer and a second primer: ′AGTGGGATCCGTTACTGATGGAGAGTTAT 3′ (SEQID NO:29)

were used to amplify the sequence found at the 5′ end of PDGF-D. In thePCR reaction a special polymerase mix was used (Advantage<<-GC cDNA PCRKit, Clontech, Cat# K1907-1). The reaction mix included (inmicroliters): Adaptor Primer 1 Gene specific primers 1 each Template(Human Heart cDNA) 5 GC-Melt (from the K1907-1 Kit) 5 5xGC cDNA PCRReaction Buffer 10 50x dNTP mix 1 Sterile H₂O 27 Total 50

The 5′ end of PDGF-D was amplified for 31 cycles, five cycles consistedof 45 seconds denaturation at 94° C. and four minutes extension at 72°C., five cycles consisted of 45 seconds denaturation at 94° C. and fourminutes extension at 70° C., and twenty-one cycles consisted of 45seconds denaturation at 94° C. and four minutes extension at 68° C. andan initial denaturation step at 94° C. for two minutes. From this PCR,an approximately 790 bp long product was obtained. This product was runon a 1% agarose gel, purified (QIAquick gel extraction Kit, Qiagen, Cat# 28706) from the gel, cloned into a vector (TOPO TA Cloning Kit,Invitrogen) and transformed into bacteria (E. Coli). Transformedbacteria were plated, and incubated at 37° C. overnight. Single colonieswere picked and grown in fresh media overnight. Plasmids were prepared(QIAprep Spin Miniprep Kit, Qiagen, Cat# 27106) and sequenced with theplasmid primers, T7 and M13R. The result of this sequencing was that 312bp of previously unknown PDGF-D sequence was obtained. The rest of thesequence (478 bp) was identical with previously obtained sequence fromother PDGF-D cDNA clones.

Similar to PDGF-C, PDGF-D has a two domain structure with a N-terminalCUB domain (residues 67-167, discussed below) and a C-terminal PDGF/VEGFhomology domain (residues 272-362, the core domain). The overall aminoacid sequence identity between PDGF-C (SEQ ID NO:32) and PDGF-D (SEQ IDNO:8) is approximately 43% (FIG. 9). The similarities are highest in thedistinct protein domains while the N-terminal region, including thehydrophobic signal sequence, and the hinge region between the twodomains display lower identities. A putative signal peptidase cleavagesite was identified between residues 22-23. Cleavage results in aprotein of 348 residue with a calculated molecular mass (M_(r)) of44,000. A single putative site for N-linked glycosylation was identifiedin the core domain of PDGF-D (residues 276-278).

FIG. 10 shows the amino acid sequence alignment of thePDGF/VEGF-homology domain of PDGF-D (found in the C-terminal region ofthe polypeptide) with the PDGF/VEGF-homology domains of PDGF/VEGF familymembers, PDGF-C, PDGF-A, PDGF-B, VEGF₁₆₅, PlGF-2, VEGF-B₁₆₇, VEGF-C andVEGF-D (SEQ ID NOs:10-18, respectively). Gaps were introduced tooptimize the alignment. This alignment was generated using the MEGALIGNalignment tool based on the method of J. Hein, (1990, Methods Enzymol.183:626-45) The PAM 250 residue weight table is used with a gap penaltyof eleven and a gap length penalty of three and a K-tuple value of twoin the pairwise alignments. The alignment is then refined manually, andthe number of identities are estimated in the regions available for acomparison.

The alignment shows that the core domain of PDGF-D displays about a 50%identity to the corresponding domain in PDGF-C, and about a 20-23%identity to the core domains in the classical PDGFs and VEGFs. It alsoshows that, with two exceptions, PDGF-D has the expected pattern ofinvariant cysteine residues, involved in inter- and intra-disulfidebonding, a hallmark of members of this family. The first exceptionoccurs between cysteine 3 and 4. Normally these two cysteines are spacedby 2 residues. However, similar to PDGF-C, PDGF-D has an uniqueinsertion of three additional amino acids residues, NCG. In total, tencysteine residues reside in the core domain, including the extremeC-terminal region, suggesting a unique arrangement of the cysteines inthe disulfide-bonded PDGF-D dimer. The second exception is that theinvariant fifth cysteine found in the other members of the PDGF/VEGFfamily is not conserved in PDGF-D. This feature is unique to PDGF-D.

Based on the amino acid sequence alignments in FIG. 10, a phylogenetictree was constructed and is shown in FIG. 11. The data show that thePDGF/VEGF homology domain of PDGF-D forms a subgroup of the PDGFstogether with PDGF-C.

CUB Domain

The N-terminal region of the partial PDGF-D amino acid sequence of FIG.12 (residues 53-170 of SEQ ID NO:8) has a second distinct protein domainwhich is referred to as a CUB domain (Bork and Beckmann, 1993, J. Mol.Biol. 231:539-545). This domain of about 115 amino acids was originallyidentified in complement factors C1r/C1s, but has recently beenidentified in several other extracellular proteins including signalingmolecules such as bone morphogenic protein 1 (BMP-1) (Wozney et al.,1988, Science, 242:1528-1534) as well as in several receptor moleculessuch as neuropilin-1 (NP-1) (Soker et al., 1998, Cell 92:735-745). Thefunctional roles of CUB domains are not clear but they may participatein protein-protein interactions or in interactions with carbohydratesincluding heparin sulfate proteoglycans. These interactions may play arole in the proteolytic activation of PDGF-D.

As shown in FIG. 12, the amino acid sequences from severalCUB-containing proteins were aligned. The results show that the singleCUB domain in human PDGF-D (SEQ ID NO:19) displays a significantidentify with the most closely related CUB domains. Sequences from humanBMP-1, with 3 CUB domains (CUBs1-3) (SEQ ID NOs: 20-22, respectively)and human neuropilin-1 with 2 CUB domains (CUBs1-2) (SEQ ID NOs: 23-24,respectively) are shown. This alignment was generated as describedabove.

EXAMPLE 1 Expression of Human PDGF-D in Baculovirus Infected Sf9 Cells

The portion of the cDNA encoding amino acid residues 24-370 of SEQ IDNO:8 was amplified by PCR using Taq DNA polymerase (Biolabs). Theforward primer used was 5′GATATCTAGAAGCAACCCCGCAGAGC 3′ (SEQ ID NO:33).This primer includes a XbaI site (underlined) for in frame cloning. Thereverse primer used was 5′ GCTCGAATTCTAAATGGTGATGGTGATGATGTCGAGGTGGTCTTGA 3′ (SEQ ID NO:34). This primer includes an EcoRI site(underlined) and sequences coding for a C-terminal 6× His tag precededby an enterokinase site. The PCR product was digested with XbaI andEcoRI and subsequently cloned into the baculovirus expression vector,pAcGP67A. Verification of the correct sequence of the cloned PCR productwas done by nucleotide sequencing. The expression vectors were thenco-transfected with BaculoGold linearized baculovirus DNA into Sf9insect cells according to the manufacturer's protocol (Pharmingen).Recombined baculovirus were amplified several times before beginninglarge scale protein production and protein purification according to themanual (Pharmingen).

Sf9 cells, adapted to serum free medium, were infected with recombinantbaculovirus at a multiplicity of infection of about seven. Mediacontaining the recombinant proteins were harvested four days afterinfection and were incubated with Ni-NTA-Agarose beads(Qiagen). Thebeads were collected in a column and after extensive washing with 50 mMsodium phosphate buffer pH 8, containing 300 mM NaCl (the washingbuffer), the bound proteins were eluted with increasing concentrationsof imidazole (from 100 mM to 500 mM) in the washing buffer. The elutedproteins were analyzed by SDS-PAGE using 12.5% polyacrylamide gels underreducing and non-reducing conditions.

FIG. 13 shows the results of the SDS-PAGE analysis of human recombinantPDGF-D under reducing (R) and non-reducing (NR) conditions. PDGF-D wasvisualized by staining with Coomassie Brilliant Blue. FIG. 13 also showsthat the recombinant PDGF-D migrates as a 90 kDa species undernon-reducing conditions and as a 55 kDa species under reducingconditions. This indicates that the protein was expressed as adisulfide-linked homodimer.

EXAMPLE 2 Generation of Antibodies to Human PDGF-D

Rabbit antisera against full-length PDGF-DD and against a syntheticpeptide derived from the PDGF-D sequence (residues 254-272, amino acidsequence RKSKVDLDRLNDDAKRYSC of SEQ ID NO:36 were generated. Thesepeptides were each conjugated to the carrier protein keyhole limpethemocyanin (KLH, Calbiochem) using N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) (Pharmacia Inc.) according to theinstructions of the supplier. 200-300 micrograms of the conjugates inphosphate buffered saline (PBS) were separately emulsified in FreundsComplete Adjuvant and injected subcutaneously at multiple sites inrabbits. The rabbits were boostered subcutaneously at biweekly intervalswith the same amount of the conjugates emulsified in Freunds IncompleteAdjuvant. Blood was drawn and collected from the rabbits. The sera wereprepared using standard procedures known to those skilled in the art.The antibodies to full-length PDGF-DD were affinity-purified on a columnof purified PDGF-DD coupled to CNBr-activated Sepharose 4B (Pharmacia).

As seen in FIG. 14, the antibodies did not cross-react with PDGF-C inthe immunoblot analysis. For immunoblotting analyses, the proteins wereelectrotransferred onto Hybond filters for 45 minutes.

EXAMPLE 3 Expression of PDGF-D Transcripts

To investigate the tissue expression of PDGF-D in several human tissues,a Northern blot was done using a commercial Multiple Tissue Northernblot (MTN, Clontech). The blots were hybridized at according to theinstructions from the supplier using ExpressHyb solution at 68° C. forone hour (high stringency conditions), and probed sequentially with a³²P-labeled 327 bp PCR-generated probe from the human fetal lung cDNAlibrary (see description above) and full-length PDGF-B cDNA. The blotswere subsequently washed at 50° C. in 2×SSC with 0.05% SDS for 30minutes and at 50° C. in 0.1×SSC with 0.1% SDS for an additional 40minutes. The blots were then put on film and exposed at −70° C. As shownin FIG. 15, upper panel, the highest expression of a major 4.4 kilobase(kb) transcript occurred in heart, pancreas and ovary while lowerexpression levels were noted in several other tissues includingplacenta, liver, kidney, prostate, testis, small intestine, spleen andcolon. No expression was detected in brain, lung, or skeletal muscle. Incomparison, the 3.5 kb PDGF-B transcript was abundantly expressed inheart and placenta, whereas lower levels were observed in all othertissues (FIG. 15, lower panel). Prominent co-expression of PDGF-D andPDGF-B occurred in heart, pancreas and ovary.

EXAMPLE 4 Immunohistochemistry Localization of VEGF-D in Mouse Embryos

The spatial and temporal patterns of expression of the PDGF-D protein inmouse embryos were determined by immunohistochemistry using standardprocedures and employing affinity-purified rabbit antibodies tofull-length PDGF-DD generated in Example 2 on tissue sections of embryosduring midgestation (embryonic day (E) 14.5). The embryos were fixed in4% paraformaldehyde overnight at 4° C. and processed for cryosectioning.14 μm cryosections were used for the stainings. Paraffin-embeddedsections which were prepared by routine procedures were also used. Aftersectioning, the slides were air dried for one to three hours followed bya ten minute post fixation with 4% paraformaldehyde. After washing 3×5minutes with phosphate buffered saline (PBS) containing 0.3% TritonX-100 (PBS-T), the slides were incubated in 0.3% H₂0₂ in PBS-T for 30minutes to quench the endogenous peroxidase activity. This was followedby washing 2×5 minutes with PBS-T and 2×5 minutes in PBS. Blocking ofnon-specific binding was done using 3% bovine serum albumin (BSA) in PBSfor 30 minutes. The slides were incubated with the affinity purifiedantibody to human PDGF-DD (3-9 mg of Ig/ml) overnight at 4° C. Afterwashing, the slides were incubated with the secondary Ig (goatanti-rabbit HRP, Vector Laboratories) at a dilution of 1:200 for onehour. After washing, the slides were incubated with the AB complex(Vector Laboratories) for one hour and washed with Tris pH 7.4. Either3,3′-diaminobenzidine tetrahydrochloride (DAB from SIGMA) or3-amino-9-ethyl carbazole (AEC from Vector Laboratories) was used forcolor development. The reaction was quenched by washing in Tris-HClbuffer. In control experiments the antibodies were preincubated with a30× molar excess of full-length PDGF-DD. This blocked the staining,while a similar preincubation with full-length PDGF-CC did not affectthe staining of the tissue sections. The photomicrographs were takenusing a Zeiss microscope equipped with differential interferencecontrast optics.

Intense staining for PDGF-D was noted in the developing heart, lung,kidney and some muscle derivatives. FIGS. 16-18 show the staining of theembryonic kidney. Intense staining of the highly vascularized fibrouscapsule (fc) surrounding the kidney, the adjacent adrenal gland (ag),and in the most peripheral aspect of the metanephric mesenchyme (mm) ofthe cortex was observed (FIGS. 16 and 17). Staining was also observed incells located in the basal aspect of the branching ureter (FIG. 18),while the developing nephron, including the ureter buds, glomeruli (gl)and Henle's loops, were negative. Previous analysis have shown thatPDGFR-beta is expressed by the metanephric mesenchyme and the developingvascular smooth muscle cells and mesangial cells of the developing renalcortex. In contrast, renal expression of PDGF-B is restricted toendothelial cells (Lindahl, P. et al., 1998, Development 125:3313-3322).The non-overlapping patterns of expression of the two PDGFR-beta ligandssuggests that PDGF-B and PDGF-D provide distinct signals to PDGFR-betaexpressing perivascular cells. This differential localization indicatesthat PDGF-D might have a paracrine role in the proliferation and/orcommitment of PDGFR-beta expressing perivascular progenitor cells of theundifferentiated metanephric mesenchyme. In line with the phenotype ofPDGF-B deficient mice, PDGF-B may then provide proliferative signals andspatial clues of the branching vascular tree of the kidney, thusallowing proliferation and co-recruitment of the PDGFR-beta expressingperivascular cells to form the mesangium of the glomeruli, and thesmooth muscle cells of the efferent and afferent arterioles.

The expression of PDGF-D partially overlaps with the expression ofPDGF-C in the cortical area of the developing kidney. The differentreceptor specificities of PDGF-C and PDGF-D and their apparent inabilityto form heterodimers indicate that the two novel PDGFs may providedistinct signals for migration and proliferation for at least twodifferent cell populations in the undifferentiated metanephricmesenchyme; either interstitial cell progenitors expressing PDGFalpha-receptor, or the PDGFR-beta expressing perivascular progenitorcells.

The phenotypic differences in the kidneys of mice lacking PDGFR-alphaand PDGF-A argue for a unique role of PDGF-C in the formation of therenal mesenchyme. Interestingly, a comparison of the PDGFR-beta andPDGF-B deficient mice have not revealed a similar phenotypic discrepancyarguing for, at least partially, redundant roles of PDGF-D and PDGF-Bduring early stages of kidney development.

EXAMPLE 5 Receptor Binding Properties of PDGF-D with the VEGF Receptors

To assess the interactions between PDGF-D and the VEGF receptors,truncated PDGF-D was tested for its capacity to bind to solubleIg-fusion proteins containing the extracellular domains of humanVEGFR-1, VEGFR-2 and VEGFR-3 (Olofsson et al., 1998, Proc. Natl. Acad.Sci. USA 95:11709-11714). An expression vector encoding the PDGF/VEGFhomology domain of PDGF-D was generated in the vector pSecTag(Invitrogen). The primers 5′-CCCAAGCTTGAAGATCTTGAGAATAT 3′ (forward)(SEQ ID NO:30) and 5′-TGCTCTAGATCGAGGTGGTCTT 3′ (reverse) (SEQ ID NO:31)were used to amplify a 429 bp fragment (nucleotides 556 to 966 in FIG.5) (SEQ ID NO:5) encoding amino acid residues 186 to 322 of FIG. 6 (SEQID NO:6). The fragment was subsequently cloned into a HindIII and XbaIdigested expression vector. COS cells were transfected with theexpression vector encoding truncated PDGF-D or a control vector usingcalcium phosphate precipitation. The expressed polypeptide included aC-terminal c-myc tag and a 6× His tag (both derived from the pSecTagvector).

The Ig-fusion proteins, designated VEGFR-1-Ig, VEGFR-2-Ig andVEGFR-3-Ig, were transiently expressed in human 293 EBNA cells. AllIg-fusion proteins were human VEGFRs. Cells were incubated for 24 hoursafter transfection, washed with Dulbecco's Modified Eagle Medium (DMEM)containing 0.2% bovine serum albumin (BSA) and starved for 24 hours. Thefusion proteins were then precipitated from the clarified conditionedmedium using protein A-Sepharose beads (Pharmacia). The beads werecombined with 100 microliters of 10× binding buffer (5% BSA, 0.2% Tween20 and 10 μg/ml heparin) and 900 microliter of conditioned mediumprepared from the COS cells transfected with the expression vector fortruncated PDGF-D or the control vector. The cells were thenmetabolically labeled with ³⁵S-cysteine and methionine (Promix,Amersham) for 4 to 6 hours. After 2.5 hours at room temperature, theSepharose beads were washed three times with binding buffer at 4° C.,once with phosphate buffered saline (PBS) and boiled in SDS-PAGE buffer.Labeled proteins that were bound to the Ig-fusion proteins were analyzedby SDS-PAGE under reducing conditions. Radiolabeled proteins weredetected using a phosphorimager analyzer and/or on film. In all theseanalyses, radiolabeled PDGF-D failed to show any interaction with any ofthe VEGF receptors. These results indicate that secreted truncatedPDGF-D does not bind to VEGF receptors R1, R2 and R3.

EXAMPLE 6 PDGFR-Beta Phosphorylation

To test if PDGF-D causes increased phosphorylation of the PDGFR-beta,full-length and plasmin-digested PDGF-D were tested for their capacityto bind to the PDGFR-beta and stimulate increased phosphorylation.

A plasmin-digested preparation of PDGF-DD was generated and analyzedsince it is known that plasmin-digestion of full-length PDGF-CC releasesthe core domain and thus allow the ligand to interact with the receptor.Full length PDGF-DD was digested with plasmin in 20 mM Tris-HCl (pH 7.5)containing 1 mM CaCl₂, 1 mM MgCl₂ and 0.01% Tween 20 for 1.5 to 4.5hours at 37° C. using two to three units of plasmin (Sigma) per ml.

Analysis of the plasmin-digested preparation of PDGF-DD by SDS-PAGEunder reducing conditions showed two prominent bands of 28 kDa and 15kDa. The 15 kDa band was identified as the core domain due to itsimmunoreactivity in immunoblotting with a peptide antiserum raisedagainst a sequence of PDGF-D just N-terminal of the first cysteineresidue in the core domain.

Serum-starved porcine aortic endothelial-1 (PAE-1) cells stablyexpressing the human PDGFR-beta (Eriksson et al., 1992, EMBO J.11:543-550) were incubated on ice for 90 minutes with a solution ofconditioned media mixed with an equal volume of PBS supplemented with 1mg/ml BSA and 10 ng/ml of PDGF-BB, 300 ng/ml or 1200 ng/ml of fulllength human PDGF-DD homodimers or 300 ng/ml or 1200 ng/ml of digestedPDGF-DD. The full length and digested PDGF-DD homodimers were producedas described above. Sixty minutes after the addition of thepolypeptides, the cells were lysed in lysis buffer (20 mM tris-HCl, pH7.5, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mM EDTA, 1 mMorthovanadate, 1 mM PMSF 1% Trasylol). The PDGFR-beta wereimmunoprecipitated from cleared lysates with rabbit antisera against thehuman PDGFR-beta (Eriksson et al., 1992, supra). The precipitatedreceptors were applied to a SDS-PAGE gel. After SDS gel electrophoresis,the precipitated receptors were transferred to nitrocellulose filters,and the filters were probed with anti-phosphotyrosine antibody PY-20,(Transduction Laboratories). The filters were then incubated withhorseradish peroxidase-conjugated anti-mouse antibodies. Boundantibodies were detected using enhanced chemiluminescence (ECL, AmershamInc). The filters were then stripped and reprobed with the PDGFR-betarabbit antisera, and the amount of receptors was determined byincubation with horseradish peroxidase-conjugated anti-rabbitantibodies. Bound antibodies were detected using enhancedchemiluminescence (ECL, Amersham Inc). The probing of the filters withPDGFR-beta antibodies confirmed that equal amounts of the receptor werepresent in all lanes. Human recombinant PDGF-BB (100 ng/ml) anduntreated cells were included in the experiment as a control. FIG. 19shows plasmin-digested PDGF-DD efficiently induced PDGFR-beta tyrosinephosphorylation. Full-length PDGF-DD failed to induce PDGFR-betatyrosine phosphorylation. PDGF-BB was included in the experiment as apositive control. This indicates that plasmin-digested PDGF-D is aPDGFR-beta ligand/agonist.

EXAMPLE 7 Competitive Binding Assay

Next, full length and plasmin-digested PDGF-D were tested for theircapacity to bind to human PDGF alpha- and beta-receptors by analyzingtheir abilities to compete with PDGF-BB for binding to the PDGFreceptors. The binding experiments were performed on porcine aorticendothelial-1 (PAE-1) cells stably expressing the human PDGF alpha- andbeta-receptors, respectively (Eriksson et al., 1992, supra). Bindingexperiments were performed essentially as in Heldin et al. (1998, EMBOJ. 7:1387-1393). Different concentrations of human full-length andplasmin-digested PDGF-DD, or human PDGF-BB were mixed with 5 ng/ml of¹²⁵I-PDGF-BB in binding buffer (PBS containing 1 mg/ml of bovine serumalbumin). Aliquots were incubated with the receptor expressing PAE-1cells plated in 24-well culture dishes on ice for 90 minutes. Afterthree washes with binding buffer, cell-bound ¹²⁵I-PDGF-BB or¹²⁵I-PDGF-AA was extracted by lysis of cells in 20 mM Tris-HCl, pH 7.5,10% glycerol, 1% Triton X-100. The amount of cell bound radioactivitywas determined in a gamma-counter. An increasing excess of the unlabeledprotein added to the incubations competed efficiently with cellassociation of the radiolabeled tracer.

FIG. 20 provides a graphical representation of results which show thatconditioned medium containing plasmin-digested PDGF-DD competes forbinding with PDGF-BB homodimers for the PDGFRs-beta, while the fulllength protein did not. Compared to PDGF-BB, plasmin-activated PDGF-DDappeared 10-12 fold less efficient as a competitor; probably a result ofsuboptimal activation of the recombinant protein in vitro by theprotease. Control experiments showed that plasmin present in thedigested PDGF-DD fraction did not affect the binding of ¹²⁵I-labelledPDGF-BB to the PDGFR-β-expressing cells. Both the full length andplasmin-digested PDGF-DD proteins failed to compete for binding to thePDGFR-alpha (FIG. 21).

These studies indicate that PDGF-DD is a PDGFR-beta-specific agonist andthat proteolytic processing releases the core domains of PDGF-DD fromthe N-terminal CUB domains which is necessary for unmasking thereceptor-binding epitopes of the core domain similar to the situationfor PDGF-CC.

EXAMPLE 8 Determination of Alternative Splicing of Murine PDGF-D

Primers were designed for the amplification of the whole coding area ofmurine PDGF-D by PCR from mouse heart cDNA (Clontech). These primerswere: 5′-CAAATGCAACGGCTCGTTT-3′ (SEQ ID NO:41) and5′-GATATTTGCTTCTTCTTGCCATGG-3′ (SEQ ID NO:42). PCR reaction conditionswere as follows: PCR Cycles: 94° C. for 2 minutes, followed by 30cycles: 94° C. for 45 seconds, 62° C. for 45 seconds, 72° C. for 90seconds, and 72° C. for 7 minutes.

The expected product from this reaction was a 1.2 kb cDNA fragment.However, the product was two bands, one approximately 1.2 kb and theother only 1.0 kb. These two products were checked in a 1% agarose gel,purified from the gel (QIAquick Gel Extraction Kit, Qiagen, Cat #28706), cloned into a vector (TOPO TA Cloning Kit, Invitrogen), andtransformed into E. Coli bacteria.

Transformed bacteria were plated and incubated at 37° C. overnight. Thenext morning some single colonies were picked and grown in fresh mediumovernight. Plasmids were prepared (QIAprep Spin Miniprep Kit, Qiagen,Cat # 27106) and sequenced with plasmid primers T7 and M13R, and alsowith mPDGF-D specific primers. The results revealed three differenttypes of murine PDGF-D cDNAs, one being completely identical with theearlier mouse clones, depicted in SEQ ID NO: 35.

The second clone was almost identical to the earlier mouse sequence,however, it lacked six amino acid residues (aa 42-47) from the regionbetween the signal sequence and the CUB domain. The second clone isdepicted in SEQ ID NO:37. The third clone was comprised of part of theearlier mouse sequence, lacking amino acids 42-47 as in the secondclone, and also lacking the PDGF-homology domain. The third clone isdepicted in SEQ ID NO:39. The similarities and differences betweenregions of the three clones are depicted in FIG. 22.

The surprising results show that at least two alternatively splicedversions of the PDGF-D gene are transcribed into polyadenylated RNA. Thevariant transcript structures suggest an alternative splice acceptorsite is used in exon two, producing a variant protein lacking six aminoacid residues (ESNHLT).

In addition to lacking the above noted six amino acid residues, thethird clone also lacks the PDGF-homology domain. This is because of theskipping of exon six and the resulting frameshift. This ends the openreading frame in a stop codon after four additional amino acid residues(GIEV). As shown in detail in FIG. 23, this splice variant only containsthe amino terminal CUB domain and could potentially provide an inhibitorof PDGF-D functions. The potential inhibition function is because theactivation of full-length PDGF-D binding to the PDGFR-D requiresproteolytic removal of the CUB domain.

EXAMPLE 9 Generation of Recombinant Human PDGF-DD Core Domain

The process as described (Bergsten et al., 2001, Nat. Cell Biol.3:512-516) was followed to generate recombinant human PDGF-DD coredomain. Human PDGF-DD was expressed as a mutant full-length formcontaining a factor Xa protease cleavage site that allowed thegeneration of the active C-terminal fragment of the protein(PDGF-homology domain) upon cleavage with factor Xa. The recombinantprotein has an extreme C-terminal His₆-tag to allow its purification ona nickel-containing resin. Following purification, the protein solutionwas dialyzed against 0.1M acetic acid and lyophilized. SDS-PAGE analysisunder reducing conditions on the purified protein revealed that itmigrated as a homogenous 21 kDa species (FIG. 24). The purified proteinwas lyophilized for storage.

EXAMPLE 10 Comparison of Angiogenic Activities of the Human PDGF-DD CoreDomain with Other PDGF Isoforms

The mouse corneal micropocket assay was performed according toprocedures described in Cao et al., 1998, Proc Natl Acad Sci USA95:14389-94; Cao et al., 1999, Nature 398:381. Specifically, lyophilizedproteins were dissolved in phosphate buffer solutions (PBS) and used tomake protein bound polymer beads, as described.

The beads were then implanted in mouse cornea. Male 5-6 week-oldC57BI6/J mice were acclimated and caged in groups of six or less.Animals were anaesthetized by injection of a mixture of dormicum andhypnorm (1:1) before all procedures. Corneal micropockets were createdwith a modified von Graefe cataract knife in both eyes of each male5-6-week-old C57BI6/J mouse. A micropellet (0.35×0.35 mm) of sucrosealuminum sulfate (Bukh Meditec, Copenhagen, Denmark) coated withslow-release hydron polymer type NCC (IFN Sciences, New Brunswick, N.J.)containing various amounts of homodimers of truncated PDGF-DD wassurgically implanted into each cornal pocket.

For comparison purposes corresponding amounts of PDGF-AA, PDGF-AB,PDGF-BB, and PDGF-CC were similarly implanted into corneal pockets oftest mice. In each case, the pellet was positioned 0.6-0.8 mm from thecorneal limbus. After implantation, erythromycin/ophthalmic ointment wasapplied to each eye.

On day 5 after growth factor implantation, animals were sacrificed witha lethal dose of CO₂, and corneal neovascularization was measured andphotographed with a slit-lamp stereomicroscope. In FIG. 25A-E, arrowspoint to the implanted pellets. Vessel length and clock hours ofcircumferential neovascularization were measured. Quantitation ofcorneal neovascularization is presented as maximal vessel length (FIG.25F), clock hours of circumferential neovascnlarization (FIG. 25G), andarea of neovascularization (FIG. 25H). Graphs represent mean values (ÅSEM) of 11-16 eyes (6-8 mice) in each group.

The corneal angiogenesis model is one of the most rigorous mammalianangiogenesis models that requires a putative compound to be sufficientlypotent in order to induce neovascularization in the corneal avasculartissue. Potent angiogenic factors including FGF-2 and VEGF have profoundeffects in this system.

The results are shown in FIG. 25. The assays were done using PDGF-AA(FIG. 25A), PDGF-AB (FIG. 25B), PDGF-BB (FIG. 25C), PDGF-CC (FIG. 25D),and PDGF-DD (FIG. 25C). FIGS. 25F-H show the quantitative analysis ofvessel length, clock hours, and vessel areas (means±SD, n=4-6).

The overall angiogenic response induced by PDGF-DD was similar to thatinduced by other PDGF isoforms. The results again clearly demonstratethat the truncated PDGF-D homodimer exhibits marked angiogenic activityin vivo. In light of the foregoing test results, which demonstrate thein vivo angiogenesis inducing activity of PDGF-DD, treatments withPDGF-DD alone, or in combination with other angiogenic factors such asVEGF family members and FGFs, provide an attractive approach fortherapeutic angiogenesis of ischemic heart, brain and limb disorders.

EXAMPLE 11 PDGF-D Promotes Connective Tissue Growth During Wound Healing

The healing of wounds is a complex process involving three discreet butoverlapping stages: inflammation, proliferation and repair, andremodeling. Wound healing involves many growth factors, some of whichexert different effects on multiple cell types. PDGF in general has beenknown to be active in all stages of the healing process and to promotewound healing. It is synthesized in significant quantities throughoutthe process by a number of different cells, including platelets,macrophages, fibroblasts and endothelial cells. Despite the criticalrole played by other growth factors involved in wound healing (EGF, FGF,insulin-like growth factors, and the TGFs), only PDGF has been shown toaugment wound healing in vivo (Steed, 1998, Am. J. Surg. 176:205-255).In fact, as early as 1991, PDGF-B was used for would healing purposes.See e.g. Pierce et al., 1991, J. Biol. Chem. 45:319-326 “Role of PDGF inwound-healing” and Pierce et al., 1994, Tissue repair processes inhealing chronic pressure ulcers treated with recombinant PDGF-BB, Am. J.Pathology 145:1399-1410. Nagai and Embil (2002) Expert Opin. Biol. Ther.2:211-218 reviewed the use of recombinantly produced PDGF-B andconcluded that it is safe, effective and easy to use in the treatmentfor healing diabetic foot ulcers.

PDGF-B and PDGF-D share the same type of receptors. The effects ofPDGF-D on wound healing were investigated using transgenic mice whichoverexpress PDGF-D in skin keratinocytes. The human PDGF-D gene wascloned and operatively linked with the keratin 14 promoter (K-14promoter), which directs the expression of the gene to the basalepithelial cells of the skin of transgenic animals (Jeltsch et al.,1997, Hyperplasia of lymphatic vessels in VEGF-C transgenic mice,Science 276:1423-1425; Detmar et al., 1998, Increased microvasculardensity and enhanced leukocyte rolling and adhesion in the skin of VEGFtransgenic mice, J. Investigative Dermatol. 111:1-6). A schematicdiagram of the K14-PDGF-D construct is depicted in FIG. 26.

Transgenic mice overexpressing PDGF-D in skin keratinocytes wereobtained. Four mice from the same transgenic litter were testedpostitive for PDGF-D and four negative. FIG. 27 shows a comparison ofPDGF-D expression between K14-PDGF-D transgenic mouse (TG) and wild-typemouse (wt). Paraffin embedded mouse skin samples were stained withanti-PDGF-D. For experimental details, see Uutela et al., 2001,Chromosomal location, exon structure and vascular expression patterns ofthe human PDGFC and PDGFD genes, Circulation 103:2242-2247, which isincorporated herein by reference in its entirety.

Mice were then anesthesised (ksylatsine+ketaminehydrochloride) andpunchbiopsy wounds were made to their flank skin (4 wounds with adiameter of 6 mm per mouse). An analgesic was used to inhibit pain(buprenorfine).

One positive and one negative mouse were sacrificed two days after thewounding, and as can be seen from FIG. 28, the amount of granulationtissue in the wound area was considerably greater in the PDGF-D positivemouse (TG) when compared with transgene negative littermate (wt).

The next mice were sacrificed after 4 days. The amount of the developingconnective tissue was greater in PDGF-D expressing mouse as shown by thevan Gieson elastic connnective tissue stain (FIG. 29). A similaraugmentation of connective tissue development was seen in the transgenicmice sacrificed 7 and 10 days after wounding (FIG. 30).

Because increased amount of elastic connective tissue results in agreater tensile strength of the PDGF-D treated wounds, these resultsindicate that PDGF-D enhances the wound repair process, and that PDGF-Dcan be used as a valuable enhancer of wound healing.

The ability of PDGF-D to stimulate wound healing is also tested in themost clinically relevant model available, as described in Schilling etal., 1959, Surgery 46:702-710 and utilized by Hunt et al., 1967, Surgery114:302-307.

Bioassays to Determine the Function of PDGF-D

Assays are conducted to evaluate whether PDGF-D has similar activitiesto PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C and/or VEGF-D in relation togrowth and/or motility of connective tissue cells, fibroblasts,myofibroblasts and glial cells; to endothelial cell function; toangiogenesis; and to wound healing. Further assays may also beperformed, depending on the results of receptor binding distributionstudies.

I. Mitogenicity of PDGF-D for Endothelial Cells

To test the mitogenic capacity of PDGF-D for endothelial cells, thePDGF-D polypeptide is introduced into cell culture medium containing 5%serum and applied to bovine aortic endothelial cells (BAEs) propagatedin medium containing 10% serum. The BAEs are previously seeded in24-well dishes at a density of 10,000 cells per well the day beforeaddition of the PDGF-D. Three days after addition of this polypeptidethe cells are dissociated with trypsin and counted. Purified VEGF isincluded in the experiment as positive control.

II. Mitogenicity of PDGF-D for Fibroblasts

To test the mitogenic capacity of PDGF-D for fibroblasts, differentconcentrations of truncated homodimers of PDGF-DD or PDGF-AA (ascontrol) are added to serum starved human foreskin fibroblasts in thepresence of 0.2 □mCi [3H]thymidine. The fibroblasts are then incubatedfor 24 hours with 1 ml of serum-free medium supplemented with 1 mg/mlBSA. After trichloroacetic acid (TCA) precipitation, the incorporationof [3H]thymidine into DNA is determined using a beta-counter. The assayis performed essentially as described in Mori et al., 1991, J. Biol.Chem. 266:21158-21164.

III. Assays of Endothelial Cell Function

a) Endothelial Cell Proliferation

Endothelial cell growth assays are performed by methods well known inthe art, e.g. those of Ferrara & Henzel, 1989, Nature 380:439-443,Gospodarowicz et al., 1989, Proc. Natl. Acad. Sci. USA 86:7311-7315,and/or Claffey et al., 1995, Biochem. Biophys. Acta 1246:1-9.

b) Cell Adhesion Assay

The effect of PDGF-D on adhesion of polymorphonuclear granulocytes toendothelial cells is tested.

c) Chemotaxis

The standard Boyden chamber chemotaxis assay is used to test the effectof PDGF-D on chemotaxis.

d) Plasminogen Activator Assay

Endothelial cells are tested for the effect of PDGF-D on plasminogenactivator and plasminogen activator inhibitor production, using themethod of Pepper et al., 1991, Biochem. Biophys. Res. Commun.181:902-906.

e) Endothelial Cell Migration Assay

The ability of PDGF-D to stimulate endothelial cells to migrate and formtubes is assayed as described in Montesano et al., 1986, Proc. Natl.Acad. Sci. USA 83:7297-7301. Alternatively, the three-dimensionalcollagen gel assay described in Joukov et al., 1996, EMBO J. 15:290-298or a gelatinized membrane in a modified Boyden chamber (Glaser et al.,1980, Nature 288:483-484) may be used.

IV. Angiogenesis Assay

The ability of PDGF-D to induce an angiogenic response in chickchorioallantoic membrane is tested as described in Leung et al., 1989,Science, 246:1306-1309. Alternatively the rat cornea assay of Rastinejadet al., 1989, Cell, 56:345-355 may be used; this is an accepted methodfor assay of in vivo angiogenesis, and the results are readilytransferrable to other in vivo systems.

V. The Hemopoietic System

A variety of in vitro and in vivo assays using specific cell populationsof the hemopoietic system are known in the art, and are outlined below.In particular a variety of in vitro murine stem cell assays usingfluorescence-activated cell sorter to purified cells are particularlyconvenient:

a) Repopulating Stem Cells

These are cells capable of repopulating the bone marrow of lethallyirradiated mice, and have the Lin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺ phenotype.PDGF-D is tested on these cells either alone, or by co-incubation withother factors, followed by measurement of cellular proliferation by³H-thymidine incorporation.

b) Late Stage Stem Cells

These are cells that have comparatively little bone marrow repopulatingability, but can generate D13 CFU-S. These cells have the Lin⁻, Rh^(h1),Ly-6A/E⁺, c-kit⁺ phenotype. PDGF-D is incubated with these cells for aperiod of time, injected into lethally irradiated recipients, and thenumber of D13 spleen colonies is enumerated.

c) Progenitor-Enriched Cells

These are cells that respond in vitro to single growth factors and havethe Lin⁻, Rh^(h1), Ly-6A/E⁺, c-kit⁺ phenotype. This assay will show ifPDGF-D can act directly on haemopoietic progenitor cells. PDGF-D isincubated with these cells in agar cultures, and the number of coloniespresent after 7-14 days is counted.

VI. Atherosclerosis

Smooth muscle cells play a crucial role in the development or initiationof atherosclerosis, requiring a change of their phenotype from acontractile to a synthetic state. Macrophages, endothelial cells, Tlymphocytes and platelets all play a role in the development ofatherosclerotic plaques by influencing the growth and phenotypicmodulations of smooth muscle cell. An in vitro assay using a modifiedRose chamber in which different cell types are seeded on to oppositecover slips measures the proliferative rate and phenotypic modulationsof smooth muscle cells in a multicellular environment, and is used toassess the effect of PDGF-D on smooth muscle cells.

VII. Metastasis

The ability of PDGF-D to inhibit metastasis is assayed using the Lewislung carcinoma model, for example using the method of Cao et al., 1995,J. Exp. Med. 182:2069-2077.

VIII. Migration of Smooth Muscle Cells

The effects of the PDGF-D on the migration of smooth muscle cells andother cells types can be assayed using the method of Koyama et al.,1992, J. Biol. Chem. 267:22806-22812.

IX. Chemotaxis

The effects of the PDGF-D on chemotaxis of fibroblast, monocytes,granulocytes and other cells can be assayed using the method of Siegbahnet al., 1990, J. Clin. Invest. 85:916-920.

X. PDGF-D in Other Cell Types

The effects of PDGF-D on proliferation, differentiation and function ofother cell types, such as liver cells, cardiac muscle and other cells,endocrine cells and osteoblasts can readily be assayed by methods knownin the art, such as ³H-thymidine uptake by in vitro cultures.

XI. Construction of PDGF-D Variants and Analogues

PDGF-D is a member of the PDGF family of growth factors which exhibits ahigh degree of homology to the other members of the PDGF family. PDGF-Dcontains seven conserved cysteine residues which are characteristic ofthis family of growth factors. These conserved cysteine residues formintra-chain disulfide bonds which produce the cysteine knot structure,and inter-chain disulfide bonds that form the protein dimers which arecharacteristic of members of the PDGF family of growth factors. PDGF-Dinteracts with a protein tyrosine kinase growth factor receptor.

In contrast to proteins where little or nothing is known about theprotein structure and active sites needed for receptor binding andconsequent activity, the design of active mutants of PDGF-D is greatlyfacilitated by the fact that a great deal is known about the activesites and important amino acids of the members of the PDGF family ofgrowth factors.

Published articles elucidating the structure/activity relationships ofmembers of the PDGF family of growth factors include for PDGF: Oestmanet al., 1991, J. Biol. Chem., 266:10073-10077; Andersson et al., 1992,J. Biol. Chem., 267:11260-1266; Oefner et al., 1992, EMBO J.,11:3921-3926; Flemming et al., 1993, Molecular and Cell Biol.,13:4066-4076 and Andersson et al., 1995, Growth Factors, 12:159-164; andfor VEGF: Kim et al., 1992, Growth Factors, 7:53-64; Pötgens et al.,1994, J. Biol. Chem., 269:32879-32885 and Claffey et al., 1995, Biochem.Biophys. Acta, 1246:1-9. From these publications it is apparent thatbecause of the eight conserved cysteine residues, the members of thePDGF family of growth factors exhibit a characteristic knotted foldingstructure and dimerization, which result in formation of three exposedloop regions at each end of the dimerized molecule, at which the activereceptor binding sites can be expected to be located.

Based on this information, a person skilled in the biotechnology artscan design PDGF-D mutants with a very high probability of retainingPDGF-D activity by conserving the eight cysteine residues responsiblefor the knotted folding arrangement and for dimerization, and also byconserving, or making only conservative amino acid substitutions in thelikely receptor sequences in the loop 1, loop 2 and loop 3 region of theprotein structure.

As used herein, the term “conservative substitution” denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative substitutions include the substitutionof one hydrophobic residue such as isoleucine, valine, leucine, alanine,cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine,norleucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic acid for aspartic acid, or glutamine for asparagine, and thelike. Neutral hydrophilic amino acids which can be substituted for oneanother include asparagine, glutamine, serine and threonine. The term“conservative substitution” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid.

As such, it should be understood that in the context of the presentinvention, a conservative substitution is recognized in the art as asubstitution of one amino acid for another amino acid that has similarproperties. Exemplary conservative substitutions are set out in thefollowing Table A from WO 97/09433. TABLE A Conservative Substitutions ISIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic Non-polar G A P I L VPolar - uncharged C S T M N Q Polar - charged D E K R Aromatic H F W YOther N Q D E

Alternatively, conservative amino acids can be grouped as described inLehninger, Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y.(1975), pp. 71-77 as set out in the following Table B. TABLE BConservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C.Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T YB. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged(Basic): K R H Negatively Charged (Acidic): D E

Exemplary conservative substitutions are set out in the following TableC. TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr(Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

If desired, the peptides of the invention can be modified, for instance,by glycosylation, amidation, carboxylation, or phosphorylation, or bythe creation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives of the peptides of theinvention. The peptides also can be modified to create peptidederivatives by forming covalent or noncovalent complexes with othermoieties. Covalently-bound complexes can be prepared by linking thechemical moieties to functional groups on the side chains of amino acidscomprising the peptides, or at the N— or C-terminus.

In particular, it is anticipated that the aforementioned peptides can beconjugated to a reporter group, including, but not limited to aradiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acolorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin).

The formation of desired mutations at specifically targeted sites in aprotein structure is considered to be a standard technique in thearsenal of the protein chemist (Kunkel et al., 1987, Methods in Enzymol.154:367-382). Examples of such site-directed mutagenesis with VEGF canbe found in Pötgens et al., 1994, J. Biol. Chem. 269:32879-32885 andClaffey et al., 1995, Biochem. Biophys. Acta, 1246:1-9. Indeed,site-directed mutagenesis is so common that kits are commerciallyavailable to facilitate such procedures (e.g. Promega 1994-1995Catalog., Pages 142-145).

The connective tissue cell, fibroblast, myofibroblast and glial cellgrowth and/or motility activity, the endothelial cell proliferationactivity, the angiogenesis activity and/or the wound healing activity ofPDGF-D mutants can be readily confirmed by well-established routinescreening procedures. For example, a procedure analogous to theendothelial cell mitotic assay described by Claffey et al., 1995,Biochem. Biophys. Acta. 1246:1-9) can be used. Similarly the effects ofPDGF-D on proliferation of other cell types, on cellular differentiationand on human metastasis can be tested using methods which are well knownin the art.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

1. An antibody specifically reactive with a polypeptide comprising acidresidues 272 to 362 of SEQ ID NO:8, or a fragment or analog thereofhaving the ability to stimulate one or more of proliferation,differentiation, motility or survival of cells expressing a PDGF-Dreceptor.
 2. The antibody according to claim 1, wherein said antibody isspecifically reactive with a polypeptide comprising amino acid residues254 to 370 of SEQ ID NO:8.
 3. The antibody according to claim 1, whereinsaid antibody is specifically reactive with a polypeptide comprising theamino acid sequence of SEQ ID NO:6.
 4. The antibody according to claim1, wherein said antibody is specifically reactive with a polypeptidecomprising amino acid residues 24 to 370 of SEQ ID NO:8.
 5. The antibodyaccording to claim 1, wherein said antibody is specifically reactivewith a polypeptide comprising the amino acid sequence of SEQ ID NO:6, orthe amino acid sequence of SEQ ID NO:8, or a fragment or analog thereofhaving the ability to stimulate one or more of proliferation,differentiation, motility or survival of cells expressing a PDGF-Dreceptor.
 6. The antibody according to claim 1, wherein said antibody isspecifically reactive with a polypeptide comprising the amino acidsequence of SEQ ID NO:6, or the amino acid sequence of SEQ ID NO:8. 7.The antibody according to claim 1, wherein said antibody is a polyclonalantibody.
 8. The antibody according to claim 1, wherein said antibody isa monoclonal antibody.
 9. The antibody according to claim 8, whereinsaid antibody is a humanized antibody.
 10. The antibody according toclaim 1, wherein said antibody is labeled with a detectable label. 11.The antibody according to claim 10, wherein said detectable label isradioactive isotope.
 12. The antibody according to claim 10, whereinsaid detectable label is an enzymatic label.
 13. The antibody accordingto claim 1, wherein the antibody is modified by addition of cytotoxic orcytostatic drug.
 14. The antibody specifically reactive with apolypeptide encoded by a polynucleotide comprising nucleotides 935 to1285 of SEQ ID NO:7, or by a polynucleotide which remains hybridizedwith the polynucleotide under a washing condition of 42° C. in 0.2×SSC.15. The antibody according to claim 14, wherein said antibody isspecifically reactive with a polypeptide encoded by at least nucleotides176 to 1285 of SEQ ID NO:7.
 16. The antibody according to claim 14,wherein said antibody is a polyclonal antibody.
 17. The antibodyaccording to claim 14, wherein said antibody is a monoclonal antibody.18. The antibody according to claim 17, wherein said antibody is ahumanized antibody.
 19. The antibody according to claim 14, wherein saidantibody is labeled with a detectable label.
 20. The antibody accordingto claim 19, wherein said detectable label is radioactive isotope. 21.The antibody according to claim 19, wherein said detectable label is anenzymatic label.
 22. The antibody according to claim 13, wherein theantibody is coupled to a cytotoxic or cytostatic compound.
 23. A methodfor inhibiting at least one of angiogenesis, lymphangiogenesis,neovascularization, connective tissue development and wound healing in amammal in need of such treatment, comprising the step of administeringan effective amount of an antibody of claim
 1. 24. The method accordingto claim 23, wherein the mammal has cancer.
 25. A method for detectingthe presence of PDGF-D polypeptide in a sample, comprising administeringthe antibody of claim 1 to the sample and detecting the specific bindingof the antibody to the PDGF-D polypeptide.
 26. The method according toclaim 25, wherein the antibody is labeled with a detectable label. 27.The method according to claim 26, wherein said detectable label isradioactive isotope.
 28. The method according to claim 26, wherein saiddetectable label is an enzymatic label.
 29. An antibody specificallyreactive with a CUB domain of PDGF-D.
 30. The antibody according toclaim 29, wherein the CUB domain comprises an amino acid sequence of SEQID NO:40.
 31. A method for binding an antibody of claim 29 to the CUBdomain of a PDGF-D polypeptide, comprising administering the antibody toa preparation containing the PDGF-D polypeptide.